Compositions and methods for invasive and non-invasive procedural skincare

ABSTRACT

Compositions and methods for skincare treatment are provided. The invention relates more generally to skin care treatment and more particularly, to compositions and methods for promoting healthy skin, skin regeneration, skin repair, skin bed preparation, and enhanced wound healing.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of Ser. No. 15/423,530 filed Feb. 2,2017 which claims the benefit of U.S. Provisional Application No.62/291,376, filed Feb. 4, 2016, and U.S. Provisional Application No.62/303,332, filed Mar. 3, 2016. Each of the aforementioned applicationsis incorporated by reference herein in its entirety, and each is herebyexpressly made a part of this specification. The aforementionedapplication is incorporated by reference herein in its entirety, and ishereby expressly made a part of this specification.

FIELD OF THE INVENTION

Compositions and methods for skincare treatment are provided, includingcompositions and methods for skin repair, promoting healthy skin, skinregeneration, and enhanced wound healing.

BACKGROUND OF THE INVENTION

The industry for invasive skin procedures such as cosmetic surgery,facial aesthetics, and medical lasers is estimated to expand to $3billion by 2017. Due to this significant industry growth, the need forskincare treatments effective at promoting skin regeneration andalleviating the negative side effects of invasive skin treatments isalso rapidly growing. These negative side effects are often a result ofslow and ineffective skin regeneration or wound healing, which producesprolonged periods of inflammation, skin sensitivity, scarring, bruising,dry skin, infection, and other unfortunate skin conditions.

Traditional skincare treatments for alleviating these negative sideeffects have primarily relied upon a variety of common over the counterremedies. These common post-procedures and over-the-counter remedieshave included skincare products such as facial and body lotions,skincare creams, petrolatum or petroleum jelly based products, butters,skin moisturizers, and a variety of other treatment products. However,these common remedies often may be primarily effective at treating oneparticular side effect, such as dry skin, and may not be meaningfullyefficacious at treating other negative side effects. Moreover, many ofthe wide range of skincare products in the market are focused ontreating the symptoms of slow wound healing, but do not enhance orpromote skin regeneration itself.

SUMMARY OF THE INVENTION

Compositions and methods for promoting healthy skin, enhanced skinregeneration, wound healing, and treating other skin conditions areprovided. These compositions preferably comprise two different peptides:a dipeptide, tripeptide, or tetrapeptide in combination with apentapeptide, hexapeptide or heptapeptide. The peptide combination iseffective in promoting healthy skin, skin regeneration, wound healing,and treating or ameliorating a variety of other skin conditions. Methodsare also provided for producing and using the compositions.

Accordingly, in a first aspect, a topical composition for promoting skinrepair is provided, comprising: one or more dipeptides, tripeptides, ortetrapeptides; and one or more pentapeptides, hexapeptides, orheptapeptides.

In a second aspect, a method for preparing a skin bed prior to adermatologic treatment is provided, comprising: applying the topicalcomposition of the first aspect to a skin bed, whereby the skin bed isprepared for a subsequent dermatological treatment, wherein theapplication of the topical composition to the skin bed prior to thedermatological treatment promotes healing of the damaged skin after thedermatological treatment.

In an embodiment of the second aspect, the topical composition of thefirst aspect is applied at least once a day to the skin bed.

In an embodiment of the second aspect, the topical composition of thefirst aspect is applied at least twice a day to the skin bed.

In an embodiment of the second aspect, the topical composition of thefirst aspect is first applied at least two weeks before thedermatological treatment.

In an embodiment of the second aspect, the topical composition of thefirst aspect is first applied at least four weeks before thedermatological treatment.

In a third aspect, a method for promoting skin repair after adermatological treatment is provided, comprising: applying the topicalcomposition of the first aspect to skin damaged by a dermatologicaltreatment, whereby healing of the damaged skin is promoted.

In an embodiment of the third aspect, the topical formulation of thefirst aspect is applied at least once a day to the skin bed.

In an embodiment of the third aspect, the topical formulation of thefirst aspect is applied at least twice a day to the skin bed.

In an embodiment of the third aspect, the topical formulation of thefirst aspect is first applied for at least two weeks after thedermatological treatment.

In an embodiment of the third aspect, the topical formulation of thefirst aspect is first applied for at least four weeks after thedermatological treatment.

In an embodiment of the third aspect, the method further comprises:applying the topical composition of the first aspect to a skin bed priorto the dermatological treatment. For example, the topical composition ofthe first aspect is applied at least once a day to the skin bed, or atleast twice a day to the skin bed, or is first applied at least twoweeks before the dermatological treatment, or is first applied at leastfour weeks before the dermatological treatment.

In an embodiment of the method of the second aspect or third aspect, orany of their respective embodiments, the dermatological treatment is alaser treatment.

In an embodiment of the method of the second aspect or third aspect, orany of their respective embodiments, the dermatological treatment is achemical peel.

In an embodiment of the method of the second aspect or third aspect, orany of their respective embodiments, the dermatological treatment is atreatment for actinic keratosis.

In an embodiment of the method of the second aspect or third aspect, orany of their respective embodiments, the dermatological treatment is atreatment for reducing signs of aging.

In a fourth aspect, a topical composition for promoting skin repair isprovided, comprising palmitoyl tripeptide-1 and palmitoylhexapeptide-12.

In an embodiment of the fourth aspect, the topical composition furthercomprises heptyl undecylenate.

In a fifth aspect, an anhydrous topical composition is providedcomprising phosphatidyl serine, oleuropein and caprylyl methicone,wherein the topical composition is has a viscosity of from 10000 cPs to25000 cPs, and wherein the anhydrous topical composition has an abilityto maintain stability over three cycles of temperature testing from −10°C. to 25° C.

In a sixth aspect, a topical composition for promoting skin repair isprovided, comprising: 82-92 wt. % of a cyclopentasiloxane, dimethiconecrosspolymer; 1-4 wt. % heptyl undecylenate; 0.01-0.06 wt. % ofpalmitoyl hexapeptide-12; 0.01-0.06 wt. % of palmitoyl tripeptide-1;0.25-1 wt. % of caprylyl methicone; 0.05-0.1 wt. % of phospatidylserine/lecithin; and 0.05-0.1 wt. % oleuropein.

In an embodiment of the sixth aspect, the topical composition comprises:2-5 wt. % of a first carrier comprising the palmitoyl hexapeptide-12,the first carrier further comprising pentaerythrityl tetraisostearate,caprylic/capric triglyceride, propylene carbonate, and stearalkoniumhectorite, wherein a concentration of the palmitoyl hexapeptide-12 inthe carrier is 100 ppm; 2-5 wt. % of a second carrier comprising thepalmitoyl tripeptide-1, the second carrier further comprisingpentaerythrityl tetraisostearate, caprylic/capric triglyceride,propylene carbonate, and stearalkonium hectorite, wherein aconcentration of the palmitoyl hexapeptide-12 in the carrier is 100 ppm.

In an embodiment of the sixth aspect, the topical composition furthercomprises: 1-4 wt. % PanthenylTriacetate/Naringenin; 1-4 wt. % ArnicaMontana Extract; and 0.5-2 wt. % Dunaliella Salina Extract.

In a seventh aspect, a method for preparing a skin bed prior to adermatologic treatment is provided, comprising: applying the topicalcomposition of any of the fourth through sixth aspects to a skin bed,whereby the skin bed is prepared for a dermatological treatment whichdamages skin, such that healing of the damaged skin after thedermatological treatment is promoted.

In an embodiment of the seventh aspect, the topical composition isapplied at least once a day to the skin bed for at least two weeksbefore the dermatological treatment.

In an embodiment of the seventh aspect, the topical composition isapplied at least twice a day to the skin bed for at least four weeksbefore the dermatological treatment.

In an eighth aspect, a method for promoting skin repair after adermatological treatment is provided, comprising: applying the topicalcomposition of any of the fourth through seventh aspects to skin damagedby a dermatological treatment, whereby healing of the damaged skin ispromoted.

In an embodiment of the eighth aspect, the topical composition isapplied at least once a day to the skin bed for at least two weeksbefore the dermatological treatment.

In an embodiment of the eighth aspect, the topical composition isapplied at least twice a day to the skin bed for at least four weeksbefore the dermatological treatment.

In a ninth aspect, a method for preparing a skin bed prior to adermatologic treatment is provided, comprising: applying the topicalcomposition of any of the fourth through seventh aspects to a skin bed,whereby the skin bed is prepared for a dermatological treatment whichdamages skin; and thereafter applying the topical composition to theskin damaged by the dermatological treatment, whereby healing of thedamaged skin is promoted.

In an aspect of the ninth embodiment, the topical composition is appliedat least once a day to the skin bed for at least two weeks before thedermatological treatment.

In an aspect of the ninth embodiment, the topical composition is appliedat least twice a day to the skin bed for at least four weeks before thedermatological treatment.

In an embodiment of any of the seventh through ninth aspects, thedermatological treatment is a laser treatment.

In an embodiment of any of the seventh through ninth aspects, thedermatological treatment is a chemical peel.

In an embodiment of any of the seventh through ninth aspects, thedermatological treatment is a treatment for actinic keratosis.

In an embodiment of any of the seventh through ninth aspects, thedermatological treatment is a treatment for reducing signs of aging.

Any of the features of an embodiment of any of the aspects is applicableto all other aspects and embodiments identified herein. Moreover, any ofthe features of an embodiment of any of the aspects is independentlycombinable, partly or wholly with other embodiments described herein inany way, e.g., one, two, or three or more embodiments may be combinablein whole or in part. Further, any of the features of an embodiment ofany of the aspects may be made optional to other aspects or embodiments.

DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a schematic depiction of the mechanism by which an exemplarycombination of peptides works to stimulate and restore elastin andcollagen levels in the skin.

FIG. 2 depicts schematically a tripeptide's impact on promoting skinregeneration through increased collagen and elastin synthesis, blockingferritin release of oxidized iron, attracting healing cells such ascapillary cells and macrophages, and through re-establishing new bloodflow to the injury site.

FIG. 3 depicts schematically a hexapeptide's impact on promoting skinregeneration and wound healing through the induction of elastin andcollagen production, fibroblast proliferation, regeneration of theextracellular matrix, and fibroblast keratinocyte mobility.

FIGS. 4A-4C depict the results of human subject wound healing progressfor treatments using a dual peptide treatment versus a controltreatment, the wound comprising a 3 mm erbium CO2 laser spot on his orher forearm.

FIG. 5A provides crusting/scabbing data for a laser wound studycomparing a dual peptide treatment against Aquaphor® treatment and acontrol treatment.

FIGS. 5B-5D are photographs of wound appearance on Day 4 for threedifferent wounds, each treated with either a dual peptide (FIG. 5B), theControl treatment (FIG. 5C), or Aquaphor® (FIG. 5D).

FIGS. 6A-6B provide data pertaining to wound appearance (FIG. 6A) andcrusting/scabbing (FIG. 6B) in a study evaluating the wound healingbenefits of two weeks of pretreatment with a dual peptide prior to alaser skin procedure and treatment with either a dual peptide orAquaphor® post-procedure. FIG. 6C is a Day 9 wound image for thePost-Wound dual peptide treatment and FIG. 6D is a Day 9 wound image forthe Post-Wound Aquaphor® treatment.

FIGS. 7A-7C are photographs of a patient before laser treatment, 4 dayspost laser treatment, and 9 days post laser treatment, wherein a dualpeptide pretreatment and post treatment were utilized in connection witha laser treatment utilizing an Encore UltraPulse® ActiveFX™ CO₂ laser,manufactured by Lumenis, Inc., Santa Clara, Calif.

FIG. 8 depicts the results of a study comparing elastin mRNA forfibroblast monolayers exposed to either a dual peptide combinationcomposition or a control.

FIG. 9 is a graph showing gelatinolytic activity (peaks area/μg protein)over time for MMP-2.

FIG. 10 shows a graph showing gelatinolytic activity (peaks area/μgprotein) over time.

FIG. 11 shows VEGF concentration (pg VEGF/ng DNA) as a function of GHK(ng/mL) concentration.

FIGS. 12A-12B provide photographs (100×) of skin biopsy samples for dualpeptide treated skin (FIG. 12B) and an untreated control (FIG. 12A). Thesamples were stained with H/E, and the photographs demonstrate thatpreconditioning treatment with dual peptide results in a more organizeddistribution and arrangement of the collagen.

FIGS. 13A-13B provide photographs (100×) of skin biopsy samples for dualpeptide treated skin (FIG. 13B) and an untreated control (FIG. 13A). Thesamples were stained to target elastin (brown), and the photographsdemonstrate a significant increase in elastin protein levels associatedwith dual peptide treatment.

FIGS. 14A-14B provide photographs (100×) of skin biopsy samples for dualpeptide treated skin (FIG. 14B) and an untreated control (FIG. 14A). Thesamples were stained to target pro-collagen by IHC, and the photographsdemonstrate a significant increase in pro-collagen levels associatedwith dual peptide treatment.

FIGS. 15A-15B provide photographs (100×) of skin biopsy samples for dualpeptide treated skin three weeks after application (FIG. 15B) and abaseline sample (FIG. 15A). Upper dermal increased collagen formationover three weeks of topical application was observed, with solarelastosis pushed down by new collagen and improved epidermis appearance.

FIGS. 16A-16C provide photographs (100×) of skin biopsy samples for dualpeptide treated facial skin three weeks after application (FIG. 16B),eight weeks after application (FIG. 16C) and a baseline sample (FIG.16A). In clients who have elastin in elastotic photodamaged elastictissue, topical application of the dual peptide results in the elastinmaterial to be less clumped and markedly distributed into deeper dermallayers over the eight week period.

FIGS. 17A-17D provide photographs of skin biopsy samples for dualpeptide treated skin three weeks after application at 40× (FIG. 17B), abaseline sample at 40× (FIG. 8A), a skin biopsy sample for dual peptidetreated skin three weeks after application at 100× (FIG. 17D) and abaseline sample at 100× (FIG. 17C). Decreased MMP1 staining was observedover the three week period in the pre-auricular region.

FIGS. 18A-18B provide photographs (100×) of skin biopsy samples for dualpeptide treated skin three weeks after application (FIG. 18B) and abaseline sample (FIG. 18A). Increased decorin staining was observed overthe three week period in the pre-auricular region.

FIGS. 19A-19I provide graphs of data generated during a randomized,single-blinded trial of the Alastin Procedure Enhancement Systemcompared to standard of care following IPL and/or PDL withQ-switch-alexandrite and fractionated CO₂ laser resurfacing of the face.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The following description and examples illustrate a preferred embodimentof the present invention in detail. Those of skill in the art willrecognize that there are numerous variations and modifications of thisinvention that are encompassed by its scope. Accordingly, thedescription of a preferred embodiment should not be deemed to limit thescope of the present invention.

DEFINITIONS

The terms “pharmaceutically acceptable salts” and “a pharmaceuticallyacceptable salt thereof” as used herein are broad terms, and are to begiven their ordinary and customary meaning to a person of ordinary skillin the art (and are not to be limited to a special or customizedmeaning), and refer without limitation to salts prepared frompharmaceutically acceptable, non-toxic acids or bases. Suitablepharmaceutically acceptable salts include metallic salts, e.g., salts ofaluminum, zinc, alkali metal salts such as lithium, sodium, andpotassium salts, alkaline earth metal salts such as calcium andmagnesium salts; organic salts, e.g., salts of lysine,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine), procaine, and tris;salts of free acids and bases; inorganic salts, e.g., sulfate,hydrochloride, and hydrobromide; and other salts which are currently inwidespread pharmaceutical use and are listed in sources well known tothose of skill in the art, such as, for example, The Merck Index. Anysuitable constituent can be selected to make a salt of the therapeuticagents discussed herein, provided that it is non-toxic and does notsubstantially interfere with the desired activity. In addition to salts,pharmaceutically acceptable precursors and derivatives of the compoundscan be employed. Pharmaceutically acceptable amides, lower alkyl esters,and protected derivatives can also be suitable for use in compositionsand methods of preferred embodiments. While it may be possible toadminister the compounds of the preferred embodiments in the form ofpharmaceutically acceptable salts, it is generally preferred toadminister the compounds in neutral form.

It is understood that, in any compound described herein having one ormore chiral centers, if an absolute stereochemistry is not expresslyindicated, then each center may independently be of R-configuration orS-configuration or a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure, enantiomerically enriched, racemicmixture, diastereomerically pure, diastereomerically enriched, or astereoisomeric mixture. In addition it is understood that, in anycompound described herein having one or more double bond(s) generatinggeometrical isomers that can be defined as E or Z, each double bond mayindependently be E or Z a mixture thereof.

Likewise, it is understood that, in any compound described, alltautomeric forms are also intended to be included. For example alltautomers of phosphate groups are intended to be included. Furthermore,all tautomers of heterocyclic bases known in the art are intended to beincluded, including tautomers of natural and non-natural purine-basesand pyrimidine-bases.

It is to be understood that where compounds disclosed herein haveunfilled valencies, then the valencies are to be filled with hydrogensor isotopes thereof, e.g., hydrogen-1 (protium) and hydrogen-2(deuterium).

It is understood that the compounds described herein can be labeledisotopically. Substitution with isotopes such as deuterium may affordcertain therapeutic advantages resulting from greater metabolicstability, such as, for example, increased in vivo half-life or reduceddosage requirements. Each chemical element as represented in a compoundstructure may include any isotope of said element. For example, in acompound structure a hydrogen atom may be explicitly disclosed orunderstood to be present in the compound. At any position of thecompound that a hydrogen atom may be present, the hydrogen atom can beany isotope of hydrogen, including but not limited to hydrogen-1(protium) and hydrogen-2 (deuterium). Thus, reference herein to acompound encompasses all potential isotopic forms unless the contextclearly dictates otherwise.

It is understood that the methods and combinations described hereininclude crystalline forms (also known as polymorphs, which include thedifferent crystal packing arrangements of the same elemental compositionof a compound), amorphous phases, salts, solvates, and hydrates. In someembodiments, the compounds described herein exist in solvated forms withpharmaceutically acceptable solvents such as water, ethanol, or thelike. In other embodiments, the compounds described herein exist inunsolvated form. Solvates contain either stoichiometric ornon-stoichiometric amounts of a solvent, and may be formed during theprocess of crystallization with pharmaceutically acceptable solventssuch as water, ethanol, or the like. Hydrates are formed when thesolvent is water, or alcoholates are formed when the solvent is alcohol.In addition, the compounds provided herein can exist in unsolvated aswell as solvated forms. In general, the solvated forms are consideredequivalent to the unsolvated forms for the purposes of the compounds andmethods provided herein.

Therapeutic Uses

The compositions described herein are suitable for use in skin therapy,and more particularly, for skincare treatment, promoting skinregeneration, and promoting enhanced wound healing. The compositions aresuitable for use in any surgical procedure involving a skin puncture, anincision, or other damage to the skin (e.g., chemical damage, cryogenicdamage, mechanical damage, light damage, electrical damage, heat damage,damage resulting from deformation of the skin as in stretch marks, orthe like) where enhanced wound healing (e.g., reduced time to healing,minimizing the appearance of the resulting scar, or the like) isdesirable. The compositions are suitable for use in enhanced woundhealing (e.g., acne scars, burn scars, other skin scars, chronicnonhealing wounds). The compositions are suitable for use in conjunctionwith various dermatological or medical treatments or procedures thatimpact the dermis (e.g., phototherapy, removal of ecrine glands,radiation treatment for cancer) The compositions described herein may besuitable for skin therapy procedures that include but are not limited toaesthetic laser skin resurfacing treatments, laser hair removal, browlift surgery, chemical peels (e.g., glycolic alpha hydroxyl acid peels,trichloroacetic acid peels, phenol peels, or the like), abdominoplasty,brachioplasty, blepharoplasty, mammoplasty, mastopexy, rhytidectomy orlower rhytidectomy, rhinoplasty, thigh lift surgery, melanocytic nevi(mole) removal, dermabrasion and microdermabrasion, retinoid treatments(e.g., isotretinoin, all-trans retinoic acid treatments, or the like),hyaluronic acid injections, botulinum toxin injections, fillertreatments (e.g., wrinkle fill treatments or the like), sclerotherapy,gluteal augmentation surgery, micro-needling, tattooing, and any and allother treatments for hair removal, hair growth and regrowth (e.g., inconjunction with oral or topical minoxidil), infections (in combinationwith topical, oral, or injectable antibiotics), skin rejuvenation orresurfacing, acne removal or reduction, broken capillaries, rosacea,wrinkle reduction, pore reduction, ablation of cellulite and otherdermal lipid depositions, wart and fungus removal, thinning or removalof scars including hypertrophic scars and keloids, treatment of abnormalpigmentation (e.g., port wine stains), tattoo removal, treatment of skininconsistencies (e.g., texture, color, tone, elasticity, hydration, orthe like), and general purpose lotions, e.g., hand lotions, facelotions, body lotions, etc.

A variety of surgical treatments and procedures can be conducted thatinvolve damage to skin tissue. Ambulatory phlebectomy is an outpatientprocedure developed by dermatologic surgeons that removes superficialveins (e.g., capillaries, spider veins, or varicose veins) throughsmall, slit-like incisions in the skin. The treatment can result intemporary bruising and swelling and inflammation caused by smallsegments of vein that remain in the skin. Blepharoplasty is a surgicalprocedure that can restore a youthful appearance to the eye area. Theupper and lower eyelids are lifted and loose or excess skin and fattissue are removed from the eye area. Minor swelling and bruising canresult. Cryolipolysis, also known as “fat freezing” or by the productname CoolSculpting, is a procedure that involves the non-invasivecooling of body fat to break down fat cells, resulting in a reduction ofbody fat without damage to other tissues. The effect takes severalmonths to be seen. Redness and localized bruising can result. Incryosurgery, liquid nitrogen is used to remove skin growths, fade agespots and treat early stage basal and squamous cell carcinomas. Thedoctor will apply frozen nitrogen using either a cotton swab or a spraydevice. The goal is to freeze the skin quickly and then allow it toslowly thaw to cause maximum destruction to targeted skin cells. In somecases, additional applications may be needed. When treating skin cancer,the doctor may insert a small needle containing a thermometer into thetreatment area to ensure the treated area has been sufficiently cooled.Potential complications include redness, swelling, bleeding, blisters,and healing problems. Dermabrasion is a procedure that uses a wire brushor a diamond wheel with rough edges to remove and level the upper layerof skin. The treated area heals, allowing new skin to grow in its place.It often is used for facial procedures. It can result in swelling andinfection. Dermal fillers—Juvederm, Restylane, Belotero, andVoluma—contain some form of hyaluronic acid, a substance that naturallyoccurs throughout the body and skin, with the highest concentrations inthe fluids of the eyes and joints. Radiesse is a calcium hydroxyapatitefiller, while Sculptra contains L-poly-lactic acid. Fillers can be usedto temporarily plump lips, raise depressed scars and level wrinkledskin. First, the doctor marks the areas to be injected. Topical orinjection numbing medication may be used for added comfort. Then thefillers are injected by syringe using very fine needles. The effecttypically lasts about six months. After that, additional injections arerequired to maintain the effect. Potential side effects includeswelling, bruising, bleeding, blisters, cysts, and inflammatoryreactions. Hair transplants include punch transplanting, wherein a plugcontaining hair follicles is removed from a part of the scalp where hairis more dense and transplanted to the treatment area. Hairtransplantation has a high success rate as long as there is enough donorhair. The procedure can result in skin irritation or damage where hairis removed from or transplanted to. Laser/light therapy is anon-invasive procedure that uses light energy to repair and regeneratedamaged skin. During the procedure, patients may feel a pinch similar tothe snapping of a rubber band. Topical anesthetic or chilled gel may beapplied to prevent discomfort. Treated area may be pink or red for fourto eight hours after treatment. A slight stinging sensation, similar toa mild sunburn in the treated area can be observed, which usuallysubsides within four to six hours, and, mild swelling in the treatmentarea that usually dissipates within several days can occur. Potentialcomplications include swelling and crusting. In liposuction, a surgeonuses a cannula to remove pockets of excess fat from various parts of thebody. The cannula is inserted through small incisions made in the skin.In some procedures, the fat is loosened with water or liquefied by laserto facilitate its removal. The procedure is minimally-invasive and isusually performed under local tumescent anesthesia. After fat isremoved, the incisions are usually left open to allow for drainage. Theprocedure can result in tissue damage, skin necrosis, bruising, andswelling. In microdermabrasion, a slightly rough applicator tip isapplied to the surface of the skin of the face or neck to remove theuppermost layer of skin. This results in a smoother skin texture. Theprocedure is painless and non-invasive, but can result in skinirritation or infection. Microlipoinjection is the transfer or recyclingof fat from one body area to another. Potential complications includebruising and swelling. Tattooing, which includes micropigmentation, alsoknown as permanent cosmetics, is the process of inserting coloredpigments just beneath the skin's surface. The procedure can result ininflammation or infection. Neck lifts are a surgical option to improvethe appearance of the neck. Results are typically long-lasting. Thereare two primary types of neck lifts, including cervicoplasty whereinexcess skin is surgically removed from the neck, and platsmaplasty, aprocedure which reduces the banded appearance of the neck by removing,tightening or realigning the muscles of the neck. The procedures canresult in bruising, infection, and swelling. Neuromodulators arewrinkle-relaxing injections of botulinum toxin—commercially known asBotox Cosmetic, Dysport or Xeomin—that are used to treat wrinkles, frownlines and crow's feet. A minute amount of the neuromodulator is injecteddirectly into the underlying muscle, causing it to relax and graduallysmooth out the appearance of the overlying skin. The effects typicallylast about three months. Potential complications include bruising andsoreness. Non-ablative skin rejuvenation uses a laser to improve theappearance of wrinkles, brown spots and minor scars by creating heat inthe skin without injuring the surface of the skin. The heat generated bythe laser promotes collagen production which causes the skin to tightenand look young and healthy. Nonablative lasers often are fractionated sothey deliver heat into the skin through thousands of tiny, deep columnsknown as microthermal treatment zones with intervening normal untreatedskin. The fractional approach allows the skin to heal much faster thanif the entire area was treated. This approach lessens the recoveryperiod and reduces the number of complications that can occur. Multiplesessions are needed in most cases. The procedure can cause redness,swelling, and infection. Non-invasive body contouring treatments includelow level laser therapy, which emits cold laser energy into body tissuesthat are absorbed by fat cells, which are broken down and absorbed intothe body, and ultrasound, which uses high-intensity, focused sound wavesto disrupt fat calls, causing them to dissolve gradually over time.Radiofrequency delivers controlled energy to excess fat areas, creatingheat deep within fat cells and subsequently destroying them.Radiofrequency therapy is regularly used for body contouring indermatology. Redness and bruising can result.

In each of the foregoing therapies, some degree of skin damage canresult which may be alleviated by using the peptide compositions asdescribed herein. The peptide compositions as disclosed herein can beapplied as a pretreatment to therapy (e.g., daily for 1-31 days or more,e.g., daily for one, two, three, or four weeks, before commencingtherapy) and/or as a post-treatment after therapy (e.g., daily for 1-31days or more, e.g., daily for one, two, three, or four weeks, aftercompletion of therapy).

Actinic Keratosis

The compositions as described herein are suitable for use connectionwith the treatment of actinic keratosis or certain other forms ofcancerous skin lesions. Actinic keratoses, also called solar keratoses,are scaly, crusty growths or lesions caused by damage from the sun'sultraviolet rays. They typically appear on sun-exposed areas such as theface, bald scalp, lips, and the back of the hands, and are oftenelevated, rough in texture, and resemble warts. Most become red, butsome will be tan, pink, and/or flesh-toned. If left untreated, up to tenpercent of actinic keratoses develop into squamous cell carcinoma. Inrarer instances, actinic keratoses may also turn into basal cellcarcinomas. Almost all actinic keratoses can be eliminated if treatedearly, before they become skin cancers (benign or malignant growths ortumors). Various treatment options are available, which depend on thegrowth's characteristics and the patient's age and health.

Cryosurgery is the most commonly used treatment method when a limitednumber of actinic keratosis lesions exist. Treatment can be performed inthe physician's office, and no cutting or anesthesia is required. Liquidnitrogen, applied with a spray device or cotton-tipped applicator,freezes the growths. The lesions subsequently shrink and/or blister,become crusted and fall off. Temporary redness and swelling may occurafter treatment, and in some patients, pigment may be lost, leavingwhite spots.

When actinic keratoses are numerous and widespread, commerciallyavailable topical creams, gels and solutions for treating actinickeratoses can be employed in combination with the compositions asdescribed herein. One of the most commonly used topical medications foractinic keratoses is 5-fluorouracil (5-FU) cream or solution. 5-FU incream or lotion form is applied to the lesion areas once or twice dailyfor two to four weeks. It can be used on all affected areas. 5-FU isavailable in a variety of formulations, in concentrations ranging from0.5 percent to 5 percent. Temporary side effects include redness,swelling, and crusting. Aldara or Zyclara (imiquimod) cream, alsoemployed to treat actinic keratoses, works by stimulating the immunesystem to produce interferon, a chemical that destroys cancerous andprecancerous cells. A topical formulation, e.g., at a concentration of5%, 3.75% or 2.5%, is applied to the lesion two or three times a weekfor several weeks or months. The cream is generally well-tolerated, butsome individuals develop redness and ulcerations. actinic keratoses. Agel combining hyaluronic acid with the non-steroidal anti-inflammatorydrug diclofenac may also be effective for people whose skin isoversensitive to other topical treatments. The gel is applied twice aday for two to three months. Picato® (ingenol mebutate), available inconcentrations of 0.015 and 0.05 percent, treats actinic keratoses withjust two or three days application time, including three consecutivedays for the 0.015% concentration (used on the face and scalp) and twoconsecutive days for the more concentrated 0.05% gel (used on the trunkand extremities). Skin redness, flaking/scaling, crusting, and swellingare the most common side effects. The adverse effects of thesetreatments (redness, crusting, swelling) may be alleviated by using thepeptide compositions as described herein. The peptide compositions asdisclosed herein can be applied as a pretreatment (e.g., daily for 1-31days or more, e.g., daily for one, two, three, or four weeks, beforecommencing application of 5-FU, imiquimod cream, hyaluronicacid/diclofenac, or ingenol mebutate), and/or in the same topicalformulation as 5-FU, imiquimod cream, hyaluronic acid/diclofenac, oringenol mebutate, and/or as a post-treatment after application of 5-FU,imiquimod cream, hyaluronic acid/diclofenac, or ingenol mebutate (e.g.,daily for 1-31 days or more, e.g., daily for one, two, three, or fourweeks, after application of 5-FU, imiquimod cream, hyaluronicacid/diclofenac, or ingenol mebutate has ceased).

Photodynamic therapy is also employed for treatment of actinic keratosesas well as Bowen's Disease, a superficial form of squamous cellcarcinoma that appears as a persistent red-brown scaly patch.Photodynamic therapy is especially useful for widespread lesions on theface and scalp. A light-sensitizing agent, topical 5-aminolevulinic acidor methyl aminolevulinate, is applied to the lesions. Subsequently,those medicated areas are activated by strong blue or red light, whichselectively destroys actinic keratoses. Redness, pain and swelling canresult. After the procedure, patients must strictly avoid sunlight forat least 48 hours, as UV exposure will increase activation of themedication, and may cause severe sunburns. The adverse effects of thistreatment (redness, pain, swelling) may be alleviated by using thepeptide compositions as described herein. The peptide compositions asdisclosed herein can be applied as a pretreatment to photodynamictherapy (e.g., daily for 1-31 days or more, e.g., daily for one, two,three, or four weeks, before commencing photodynamic therapy) and/or asa post-treatment after photodynamic therapy (e.g., daily for 1-31 daysor more, e.g., daily for one, two, three, or four weeks, aftercompletion of photodynamic therapy).

Cryotherapy, 5-FU, imiquimod cream, hyaluronic acid/diclofenac, ingenolmebutate, and photodynamic therapy can be combined for treatment ofactinic keratoses, along with use of the peptide compositions disclosedherein to encourage skin repair. Treatment regimens can includecryosurgery combined with photodynamic therapy or a topical agent likeimiquimod, diclofenac, of 5-FU. The topical medications and photodynamictherapy may also be used serially every three months, six months, oryear, as determined by the physician at routine skin examinations. Thisapproach may both improve the cure rate and reduce side effects. One totwo weeks of 5-FU followed by cryosurgery can reduce the healing timefor 5-FU and decrease the likelihood of white spots followingcryosurgery.

Curettage and electrodessication can be employed to remove portions ofactinic keratoses, with electrocautery or trichloroacetic acid employedto stop bleeding, optionally under local anesthesia. The peptideformulations disclosed herein can advantageously be employed as apretreatment or post-treatment. The peptide compositions as disclosedherein can be applied as a pretreatment to curettage and/orelectrodessication (e.g., daily for 1-31 days or more, e.g., daily forone, two, three, or four weeks, before commencing curettage and/orelectrodessication) and/or as a post-treatment after curettage and/orelectrodessication (e.g., daily for 1-31 days or more, e.g., daily forone, two, three, or four weeks, after completion of curettage and/orelectrodessication).

Chemical peeling, commonly employed to reverse the signs of photoaging,is also used to remove superficial actinic keratoses on the face,especially when other techniques have not succeeded. Trichloroaceticacid and/or similar chemicals are applied directly to the skin, causingthe top skin layers to slough off. New skin generally regrows within afew weeks. This technique may require local anesthesia and can causetemporary discoloration and irritation. The peptide formulationsdisclosed herein can advantageously be employed as a pretreatment orpost-treatment. The peptide compositions as disclosed herein can beapplied as a pretreatment to chemical peeling (e.g., daily for 1-31 daysor more, e.g., daily for one, two, three, or four weeks, beforecommencing chemical peeling) and/or as a post-treatment after chemicalpeeling (e.g., daily for 1-31 days or more, e.g., daily for one, two,three, or four weeks, after completion of chemical peeling).

Laser surgery can also be employed to treat actinic keratoses. Theskin's outer layer and variable amounts of deeper skin are removed usinga carbon dioxide or erbium YAG laser, as described elsewhere herein.Lasers are effective for removing actinic cheilitis from the lips andactinic keratoses from the face and scalp. They offer good control overthe depth of tissue removed. Lasers are also used as a secondary therapywhen topical medications or other techniques are unsuccessful. However,local anesthesia may be required. The risks of scarring and pigment lossare slightly greater than with other techniques. Accordingly, use ofpeptide formulations disclosed herein can advantageously be employed asa pretreatment or post-treatment. The peptide compositions as disclosedherein can be applied as a pretreatment to laser treatment (e.g., dailyfor 1-31 days or more, e.g., daily for one, two, three, or four weeks,before commencing laser treatment) and/or as a post-treatment afterlaser treatment (e.g., daily for 1-31 days or more, e.g., daily for one,two, three, or four weeks, after completion of laser treatment).

Chemical Peel

The peptide compositions described herein are useful in conjunction withchemical peel treatments. A chemical peel is a technique used to improvethe appearance of the skin on the face, neck or hands. A chemicalsolution is applied to the skin that causes it to exfoliate andeventually peel off. The new skin is usually smoother and less wrinkledthan the old skin. The new skin is also temporarily more sensitive tothe sun. There are three basic types of chemical peels. A superficialpeel can employ alpha-hydroxy acid or another mild acid to penetrateonly the outer layer of skin to gently exfoliate it. The treatment isused to improve the appearance of mild skin discoloration and rough skinas well as to refresh the face, neck, chest or hands. In a medium peel,glycolic or trichloroacetic acid is applied to penetrate the out andmiddle layers of skin to remove damaged skin cells. The treatment isused to improve age spots, fine lines and wrinkles, freckles andmoderate skin discoloration. It also can be used to smooth rough skinand treat some precancerous skin growths, e.g., actinic keratoses asdescribed elsewhere herein. In a deep peel, tricholoracetic acid orphenol is applied to deeply penetrate the middle layer of skin to removedamaged skin cells. The treatment removes moderate lines, age spots,freckles and shallow scars. Patients will see a dramatic improvement inskin appearance; however, the treatment can result in varying degrees ofdamage to the layers of the skin. The peptide compositions as disclosedherein can be applied as a pretreatment to chemical peeling (e.g., dailyfor 1-31 days or more, e.g., daily for one, two, three, or four weeks,before commencing chemical peeling) and/or as a post-treatment afterchemical peeling (e.g., daily for 1-31 days or more, e.g., daily forone, two, three, or four weeks, after completion of chemical peeling).

One type of chemical peel is a pulse peel, which involves application ofa glycolic acid peel followed by application of an agent to targetcancerous cells (e.g., 5-fluorouracil). The treatment can repeated fourto six times over a two week period. Preconditioning with theformulations of the embodiments can advantageously be employed inconjunction with a pulse peel, or can advantageously be employed tofacilitate healing when applied post-procedure.

Laser Resurfacing

In particular, the compositions described herein may be suitable for useprior to and/or following treatment with ablative and non-ablative laserresurfacing treatments that may be fractionated or unfractionated. Inablative laser treatments, the laser procedure removes the outer layersof skin at a specific targeted area. These procedures require a longerduration wound healing process than non-ablative laser treatments, whichdo not lead to the removal or vaporization of skin. Non-fractionatedlaser treatments act on the entire projected surface area of the treatedskin, whereas fractionated laser treatments act on evenly dividedportions of a targeted area to provide untouched regions of skin forquickened healing. As such, fractionated laser treatments have resultedin fewer side effects with lower amounts of reported scarring. See e.g.Preissig, J., Hamilton, K., Markus, R., Current laser resurfacingtechnologies: A review that delves beneath the surface, Seminars inPlastic Surgery 2012, Vol. 26(3), pp. 109-116. Ablative laser treatmentsmay include but are not limited to CO₂, Er:YAG (erbium-doped yttriumaluminum garnet), combined Erbium/CO₂, and fractional laserphotothermolysis laser resurfacing procedures. CO₂ lasers emit light atthe 10,600 nm wavelength at pulses ranging as low as 0.2 μs-80 μs withhigher intensity lasers and up to 10 ms pulses with lower intensitylasers. Er:YAG lasers emit light at the 2,940 nm wavelength and havepulses ranging from 0.25 up to 5 ms. Combination CO₂ and Er:YAG lasersrely on a combination of treatment with both types of lasers. Shorterpulses facilitate higher energy delivery and facilitate deeper ablationof skin. Non-ablative laser treatments may include diode, Erbium glass,thulium fiber, and Nd:YAG (neodymium-doped yttrium aluminum garnet)lasers. These treatments use lasers that emit light at wavelengthsranging from 1319 to 1927 nm at pulses ranging from 450 μs up to 210 ms.The peptide compositions as disclosed herein can be applied as apretreatment to laser treatment (e.g., daily for 1-31 days or more,e.g., daily for one, two, three, or four weeks, before commencing lasertreatment) and/or as a post-treatment after laser treatment (e.g., dailyfor 1-31 days or more, e.g., daily for one, two, three, or four weeks,after completion of laser treatment).

Skin Repair

The active ingredients of the compositions include two or more peptides.The first peptide in the combination is one or more dipeptides,tripeptides, and/or tetrapeptides and the second peptide in thecombination is one or more pentapeptides, hexapeptides, and/orheptapeptides. The compositions may be used in cosmetic, cosmeceuticaland general skincare compositions or provided in pharmaceuticalcompositions. Methods are also described for using compositionscomprising the dipeptide, tripeptide, or tetrapeptide and thepentapeptide, hexapeptide, or heptapeptide for the promotion of healthyskin, skin regeneration and enhanced wound healing.

In general, skin regeneration and wound healing involve complicated andpoorly understood processes that rely on the combined efforts of a largenumber of different tissues and cell types. Successful wound healing,which can be considered a type of tissue remodeling, occurs when thetissue remodeling process alleviates the inflammatory response of theinnate immune system and minimizes the scar forming process that occurswhen fibrous tissue replaces normal skin after an injury. This processoccurs most efficiently in young children but becomes less efficientwith age. Less efficient wound healing often produces unsightly,irritating, and even painful scars such as keloids or hypertrophicscars. Given that few of the patients undergoing invasive skinprocedures are children, skincare treatments that can promote woundhealing after an invasive skin procedure are particularly desirable. Thecombination of the two peptides has superior efficacy in promoting woundhealing and skin regeneration.

Additionally, the compositions help treat or prevent dermatologicconditions such as skin dryness, dullness, loss of elasticity, lack ofradiance, exaggerated lines and wrinkles, stretch marks, spider vesselsor red blotchiness. In some embodiments, the appearance of “marionette”lines, smile lines, deep nasolabial fold lines, crow's feet, finelines/wrinkles, vertical lines between the eyebrows, horizontal foreheadlines, sagging thin/frail skin, skin redness and dullness may beimproved using compositions as described herein. The compositions canalso be used in the prevention and treatment of photodamaged skin, theappearance of fine lines and wrinkles, hyperpigmentation, age spots, andaged skin. The disclosed compositions can also help increase theflexibility of the stratum corneum, increasing the content of collagenand/or glycosaminoglycans in skin, increasing moisture in skin,decreasing transcutaneous water loss, and generally increasing thequality of skin.

The compositions can also be employed in the connection with mucousmembranes, in particular the lips and the vaginal mucosa. When appliedto the vaginal mucosa, a vaginal applicator can be employed as arecommercially available. Suitable applicators can be in a form of apre-filled syringe, a tube attached to a prefilled squeezable reservoir,a prepackaged wand including a preselected amount of composition, or auniversal vaginal applicator including perforations along its length fordispensing the composition through the perforations.

Drug Delivery Uses

Certain of the compositions can be employed as a carrier for drugdelivery. The anhydrous compositions as disclosed herein areparticularly useful in this regard for the delivery of suitable for thedelivery of locally acting drugs such as antibacterial drugs,antiprotozoal drugs, antifungal drugs, antiviral drugs, spermicidalagents, prostaglandins, and steroids. Drugs suitable for deliveryinclude bromocriptine, sildenafil, oxytocin, calcitonin, luteinizinghormone-releasing hormone and analogues, insulin, human growth hormone,oxybutynin, and steroids used in hormone replacement therapy or forcontraception. Antifungal drugs include clotrimazole, econazole,miconazole, terbinafine, fluconazole, ketoconazole, and amphotericin.Antibiotics include amoxicillin, doxycycline, cephalexin, ciprofloxacin,clindamycin, metronidazole, azithromycin, sulfamethoxazole/trimethoprim,amoxicillin/clavulanate, and levofloxacin. Classes of antibioticsinclude penicillins, tetracyclines, cephalosporins, quinolones,lincomycins, macrolides, sulfonamides, glycopeptides, aminoglycosides,and carbapenems. Types of hormones include 5-alpha-reductase inhibitors,adrenal cortical steroids, corticotropin, glucocorticoids,mineralocorticoids, adrenal corticosteroid inhibitors, antiandrogens,antidiuretic hormones, antigonadotropic agents, antithyroid agents,aromatase inhibitors, calcitonin, estrogen receptor antagonists,gonadotropin-releasing hormone antagonists, growth hormone receptorblockers, growth hormones, insulin-like growth factor, parathyroidhormone and analogs, progesterone receptor modulators, prolactininhibitors, selective estrogen receptor modulators, sex hormones,androgens and anabolic steroids, contraceptives, estrogens, gonadotropinreleasing hormones, gonadotropins, progestins, sex hormone combinations,somatostatin and somatostatin analogs, synthetic ovulation stimulants,and thyroid drugs. Antiviral agents include adamantane antivirals,antiviral boosters, antiviral combinations, antiviral interferons,chemokine receptor antagonist, integrase strand transfer inhibitor,miscellaneous antivirals, neuraminidase inhibitors, NNRTIs, NS5Ainhibitors, nucleoside reverse transcriptase inhibitors (NRTIs),protease inhibitors, and purine nucleosides.

Drugs for treating skin conditions that can be employed using selectedcompositions as a delivery device include acne drugs (isotretinoin),atopic dermatitis drugs (topical steroids), herpes zoster drugs(antivirals such as valacyclovir), hives (antihistamines like loratadineor fexofenadine, omalizumab), sunburn (lidocaine), contact dermatitis(antihistamines, topical steroids), diaper rash (zinc oxide), rosacea(metronidazole, doxycycline, azelaic acid, isotretinoin, beta blockers,estrogen), athlete's foot (antifungals), and basal cell carcinoma(imiquimod, fluorouracil, vismodegib).

Types of Formulations

The peptide combinations of the embodiments can be employed in varioustypes of formulations. Topical formulations including a dipeptide,tripeptide, or tetrapeptide, and a pentapeptide, hexapeptide, orheptapeptide in combination with at least one excipient, are provided.Excipients can include a nonaqueous or aqueous carrier, and one or moreagents selected from moisturizing agents, pH adjusting agents,deodorants, fragrances, chelating agents, preservatives, emulsifiers,thickeners, solubilizing agents, penetration enhancers, anti-irritants,colorants, surfactants, beneficial agents, pharmaceutical agents, andother components as known in the art for use in connection with topicalformulations for treatment of the skin. Preferably, the formulation isan anhydrous formulation to prevent skin irritation such as water-basedirritant contact dermatitis or stinging sensation upon application todamaged skin. In another embodiment, the composition is formulated suchthat preservatives need not be employed (e.g., a preservative-freeformulation) so as to avoid skin irritation associated with certainpreservatives.

To facilitate application, the composition may be provided as anointment, an oil, a lotion, a paste, a powder, a gel, or a cream. Thecomposition may also include additional ingredients such as a protectiveagent, an emollient, an astringent, a humectant, a sun screening agent,a sun tanning agent, a UV absorbing agent, an antibiotic agent, anantifungal agent, an antiviral agent, an antiprotozoal agent, ananti-acne agent, an anesthetic agent, a steroidal anti-inflammatoryagent, a non-steroidal anti-inflammatory agent, an antipruritic agent,an additional antioxidant agent, a chemotherapeutic agent, ananti-histamine agent, a vitamin or vitamin complex, a hormone, ananti-dandruff agent, an anti-wrinkle agent, an anti-skin atrophy agent,a skin whitening agent, a cleansing agent, additional peptides,additional modified peptides, and combinations thereof. In a furtherembodiment, the composition may avoid animal or cellular-based materialsto avoid skin irritation. The composition can be applied to the dermis,or to mucous membranes.

Methods of using topical peptide formulations for promoting healthyskin, skin regeneration, and enhanced wound healing are provided. Thecompositions may also be applied to treat skin conditions such asinflammation, redness, soreness, skin sensitivity, dry skin, bruising,and similar conditions. Application of the peptide compositioncomprising a first dipeptide, tripeptide, or tetrapeptide and a secondpentapeptide, hexapeptide, or heptapeptide may also be used to preventscarring (e.g., in facelift procedures or other cosmetic proceduresinvolving a skin incision), to quicken epithelial confluence, and tolimit scabbing and crusting during wound healing. Increased collagenproduction and/or increased elastin production can also be inducedthrough the application of a composition that comprises a firstdipeptide, tripeptide, or tetrapeptide and a second pentapeptide,hexapeptide, or heptapeptide. Suitable methods for objectively measuringimprovement in skin redness and inflammation may include tristimuluscolorimetry, narrow-band reflectance spectroscopy, diffuse reflectancespectroscopy, skin reflectance spectroscopy, and/or UV photography.

Some embodiments include administering peptide compositions providedherein in topical formulations; however, other routes of administrationare also contemplated (e.g., mucosal, subdermal, oral, or the like).Contemplated routes of administration include but are not limited totopical, mucosal, and subcutaneous. Suitable liquid forms includesuspensions, emulsions, solutions, and the like. Unit dosage forms canalso be provided, e.g., individual packets with a premeasured amount ofthe formulation, configured for administration to the face or other bodypart on a predetermined schedule pre-procedure and post-procedure. Unitdosage forms configured for administration twice or three times a daypre-procedure and post-procedure are particularly preferred; however, incertain embodiments it can be desirable to configure the unit dosageform for administration once a day, four times a day, or more.

In some embodiments, the topical and other formulations typicallycomprise from about 0.001 wt. % or less to about 50 wt. % or more ofactive ingredient, such as the peptides, preferably from about 0.005,0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 wt. % to about 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, or 45 wt. %.

Compositions and formulations for topical administration can includetransdermal patches, ointments, lotions, creams, gels, drops, sprays,liquids, aerosols, and powders. Conventional pharmaceutical carriers,aqueous, powder or oily bases, thickeners and the like may be employed.In certain applications, an ointment, lotion, cream, gel or similarformulation can be provided that can be applied to the skin using thefingers. Such formulations are typically provided in a squeeze tube orbottle or a pot, or in a roll-on, wherein a ball is secured in the topof a container of the formulation, wherein the ball is permitted toroll. By rolling the ball over the skin surface, liquid in the containeris transferred to the skin in a controlled manner. An alternativedelivery mechanism includes a container with a perforated lid with amechanism for advancing an extrudable formulation through the lid. Inanother form, a gel formulation with sufficient structural integrity tomaintain its shape is provided, which is advanced up a tube and appliedto the skin (e.g., in a stick form). An advantage of the stick form isthat only the formulation contacts the skin in the application process,not the fingers or a portion of a container. A liquid or gel can also beplaced using an applicator, e.g., a wand, a sponge, a syringe, or othersuitable method.

Components of the Formulations

Peptides

Formulations comprising a combination of two or more peptides areprovided for promoting healthy skin, skin regeneration, and enhancedwound healing, e.g., in patients subject to a skin procedure such as alaser treatment, a chemical peel, dermabrasion, microneedling, and othersuch procedures, in patients subject to any other treatment or exposureresulting in damage, inflammation, or irritation to the skin (e.g.,sunburn, eczema, psoriasis, herpes lesions, shingles, allergic reaction,contact dermatitis, or the like), or in any skin condition whereinstimulation of collagen and/or elastin is beneficial. In a topicalformulation comprising the two peptide combination, a first peptide(e.g., tripeptide) is present in the composition in pure for or in aform of a carrier containing the peptide, e.g., 50 ppm (by weight) orless to 1000, 5000, 10000, 50000, 100000, 500000 ppm or more, e.g., 100ppm of the peptide. The topical formulation can contain from 0.01 wt. %or less (e.g., 0.001 wt. %) to 10 wt. % or more, e.g., 0.01 wt. % to0.02 wt. %, 0.03 wt. %, 0.04 wt. %, 0.05 wt. %, 0.1 wt. %, 1 wt. % to 5wt. % or 10 wt. % or 20 wt. % of the first peptide. The second peptide(e.g., hexapeptide) is present in the topical formulation composition inpure form or in a form of a carrier containing the peptide, e.g., 50 ppm(by weight) or less to 1000, 5000, 10000, 50000, 100000, 500000 ppm ormore, e.g., 100 ppm of the peptide, or any other suitable amount. Thetopical formulation can contain from 0.01 wt. % or less (e.g., 0.001 wt.%) to 10 wt. % or more, e.g., 0.01 wt. % to 0.02 wt. %, 0.03 wt. %, 0.04wt. %, 0.05 wt. %, 0.1 wt. %, 1 wt. % to 5 wt. % or 20 wt. % of thesecond peptide. The amount of peptide in the base can be adjusted up ordown.

In example embodiments, a weight ratio for the first peptide to thesecond peptide in a topical formulation is 1 part first peptide to 0.2to 10 parts second peptide, or 1 to 10 parts second peptide, or 1 to 8parts second peptide, or 1 to 5.5 parts second peptide. The followingnomenclature is employed herein to refer to various amino acids: Alanine(also referred to herein as “Ala” or “A”), Arginine (also referred toherein as “Arg” or “R”), Asparagine (also referred to herein as “Asn” or“N”), Aspartic acid (also referred to herein as “Asp” or “D”), Cysteine(also referred to herein as “Cys” or “C”), Glutamic acid (also referredto herein as “Glu” or “E”), Glutamine (also referred to herein as “Gln”or “Q”), Glycine (also referred to herein as “Gly” or “G”), Histidine(also referred to herein as “His” or “H”), Isoleucine (also referred toherein as “Ile” or “I”), Leucine (also referred to herein as “Leu” or“L”), Lysine (also referred to herein as “Lys” or “K”), Methionine (alsoreferred to herein as “Met” or “M”), Phenylalanine (also referred toherein as “Phe” or “F”), Proline (also referred to herein as “Pro” or“P”), Serine (also referred to herein as “Ser” or “S”), Threonine (alsoreferred to herein as “Thr” or “T”), Tryptophan (also referred to hereinas “Trp” or “W”), Tyrosine (also referred to herein as “Tyr” or “Y”),Valine (also referred to herein as “Val” or “V”).

In some embodiments, the first peptide is a dipeptide. Suitabledipeptides include but are not limited to those having the followingsequence of amino acids: KK, KP, CK, KC, KT, DF, NF, VW, YR, or TT. Inother embodiments, the first peptide is a tripeptide. Suitabletripeptides include but are not limited to those having the followingsequence of amino acids: HGG, RKR, GHK, GKH, GGH, GHG, KFK, or KPK. Insome embodiments, the first peptide is a tetrapeptide. Suitabletetrapeptides include but are not limited to those having the followingsequence of amino acids: GQPR, KTFK, AQTR, or RSRK. In some embodiments,the second peptide is a pentapeptide. Suitable pentapeptides include butare not limited to those having the following sequence of amino acids:KTTKS, YGGFX or KLAAK. In some embodiments, the second peptide is ahexapeptide. Suitable hexapeptides include but are not limited to thosehaving the following sequence of amino acids: VGVAPG or GKTTKS. In someembodiments, the second peptide is a heptapeptide. Suitableheptapeptides include but are not limited to one having an amino acidsequence RGYYLLE, or Heptapeptide-6 (a pro-sirtuin peptide). Thecompositions may include two or more peptides, e.g., two dipeptides andone pentapeptide; one tripeptide and one hexapeptide; one dipeptide, onetripeptide, and one heptapeptide, or the like, provided that thecomposition contains at least one dipeptide, tripeptide, or tetrapeptideand at least one pentapeptide, hexapeptide, or heptapeptide.

The peptide can be functionalized. For example, the peptide can befunctionalized with a fatty acid, e.g., myristoleic acid, palmitoleicacid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleicacid, linoelaidic acid, α-linolenic acid, arachidonic acid,eicosapentaenoic acid, erucic acid, docosahexaenoic acid, caprylic acid,capric acid, lauric acid, palmitic acid, stearic acid, arachidic acid,behenic acid, lignoceric acid, cerotic acid, or the like. Examplesinclude palmitoyl hexapeptide-12 (Pal-VGVAPG), palmitoyl tripeptide-1(Pal-GHK), myristoyl hexapeptide-12 (Myr-VGVAPG), myristoyl tripeptide-1(Myr-GHK). Palmitoyl or myristoyl functionalization can be desirable incertain embodiments as it exhibits enhanced penetration when compared toother fatty acids.

Some embodiments of the methods and compositions provided herein includeas a first peptide glycine-histidine-lysine (GHK). GHK is a peptidesequence that is rarely found in the class of proteins in general, butis frequently found in extracellular matrix proteins. The small size ofGHK permits it to approach membrane receptors far more easily thanlarger peptides. Further, its unique, copper-binding structure enhancescopper transport into and out of cells and promotes wound healingthrough several different but related pathways. Due to its strong copperbinding structure, GHK can be provided in the form of GHK-Cu(copper-bound GHK form).

GHK-Cu acts as an anti-inflammatory (see, e.g., Pickart, L., The humantri-peptide GHK and tissue remodeling, J. Biomater. Sci. Polymer Edn.2008, Vol. 19, pp. 969-988, 972-973; Pickart et al., The HumanTripeptide GHK-CU in Prevention of Oxidative Stress and DegenerativeConditions of Aging: Implications for Cognitive Health, Oxid. Med. CellLongev. 2012, Vol. 2012, pp. 1-8, 3) and an antioxidant. GHK-Cu acts topromote wound healing by suppressing the “acute phase response” that canproduce both inflammation and induce scarring. This biological responseprevents the invasion of bacteria, facilitates the arrival of immunecells, stems bleeding, and provides a covering for the wounded area.GHK-Cu also suppresses the acute phase response by inhibiting theproduction of molecules called cytokines. Cytokines are immune cellsignaling molecules that attract immune cells and that trigger theproduction of other molecules that promote inflammation and fibrosis(leading to the creation of scar tissue). In particular, GHK-Cusuppresses the production of cytokines including tumor necrosisfactor-alpha (TNFα), interleukin-1 (IL-1), interleukin-6 (IL-6), andtransforming growth factor-beta-1 (TGF-β1), a few of the key drivers ofinflammation and apoptotic cell death in the wound region. As TGF-β1 isan important component for the continuation of the acute phase response,GHK-Cu's suppression of TGF-β1 also acts to shorten the duration of theacute phase response once it has begun. GHK-Cu acts as an antioxidant byblocking ferritin's release of oxidizing iron, preventing furtherinflammation or microbial infection (as invading microbes need iron tosurvive).

GHK-Cu also stimulates blood vessel growth, increases collagenproduction, and regenerates the extracellular matrix. GHK-Cu acts as anattractant for cells vital to the regeneration of damaged tissues suchas capillary cells that rebuild blood vessels. It also upregulates theproduction of a variety of enzymes that remove damaged proteins whilealso rebuilding the extracellular matrix (ECM), a key external scaffoldthat is important for intercellular communication and support. Inparticular, GHK-Cu's induces the production of messenger RNAs (mRNAs)necessary for the regeneration of the ECM, namely collagen,proteoglycans, glycosaminoglycans, chondroitin sulfate, and dermatansulfate. GHK-Cu's induction of increased collagen production also playsa key role in enhancing skin regrowth. GHK-Cu further stimulates bloodflow into damaged tissues through three processes: angiogenesis,anti-coagulation and vascular dilation. First, GHK-Cu inducesangiogenesis or new blood vessel formation by increasing the productionof growth factor proteins necessary for angiogenesis such as basicfibroblast growth factor (BFGF) and vascular endothelial growth factor(VEGF). Second, GHK-Cu increases blood flow to the wounded area byexpanding the number of red blood cells (via growth in erythropoietinproduction) and by anti-coagulatory effects such as downregulating theblood clotting molecule thromboxane. Third, GHK-Cu facilitates vasculardilation through binding to the vasoconstriction protein angiotensin II,preventing angiotensin from constricting blood vessels and reducingblood flow.

GHK-Cu promotes stem cell proliferation (see, e.g., Ito et al., Is theHair Follicle Necessary for Normal Wound Healing, J. Invest. Dermatol.2008, Vol. 128, pp. 1059-1061, 1059). Wound healing studies havedemonstrated that the addition of GHK-Cu greatly enlarged the productionof hair follicles near the wound periphery in experiments with mice.Dermal hair follicles are a significant source of stem cells that areessential for dermal healing. Research into dermal hair follicles havedemonstrated that hair-bearing areas tend to heal more quickly and thatcells from various portions of the follicle may contribute to bothdermal cell and epithelial cell replacement as well.

Thus, by decreasing inflammation, acting as an antioxidant, stimulatinggrowth of new blood vessels, regenerating the extracellular matrix,enhancing collagen production, and by promoting stem cell proliferation,GHK can greatly enhance skin regeneration and promote wound healing.

Some embodiments of the methods and compositions provided herein includeas a second peptide valine-glycine-valine-alanine-proline-glycine(VGVAPG). VGVAPG is a hexapeptide that is derived from the elastinprotein (see, e.g., Blanchevoye et al., Interaction between the ElastinPeptide VGVAPG and Human Elastin Binding Protein, J. Biol. Chem. 2012,Vol. 288, pp. 1317-1328, 1317-1318). Elastin is a protein found inconnective tissue (e.g. skin) that is necessary for tissues to return totheir original shape and size after undergoing temporary expansion orcontraction. Due to the importance of elastin in providing elasticityand resilience, elastin plays a significant role in skin cell resistanceto injury and recovery from injury. The ability of skin to return to itsoriginal form after undergoing stretching or pulling relies oncross-linked elastin proteins (tropoelastin proteins in humans) thatwork to form “elastic fibers.” The disruption of the elastic fibersystem in healing wounds has been strongly linked to the production ofscar tissue (see, e.g., Rnjak-Kovacina et al., Severe Burn Injuries andthe Role of Elastin in the Design of Dermal Substitutes, Tissue Eng.Part B. Rev. 2011, pp. 81-91, 85-86). Because of these properties andothers, elastin is a key component in the effective wound healingprocess.

VGVAPG plays a role in facilitating elastin's ability to prevent skininjury and to promote skin regeneration (see, e.g., Floquet et al.,Structural Characterization of VGVAPG, an Elastin-Derived Peptide,Biopolymers (Peptide Science) 2004, Vol. 76, 266-280, 267). First, ithas been shown to demonstrate the ability to attract monocytes andfibroblasts (see, e.g., Senior et al., Val-Gly-Val-Ala-Pro-Gly, aRepeating Peptide in Elastin, Is Chemotactic for Fibroblasts andMonocytes, J. Cell Biol. 1984, Vol. 99, pp. 870-874, 870), monocytesbeing essential for fighting off infection and fibroblasts beingnecessary for collagen production (the most abundant protein in skin)and for the regeneration of the extracellular matrix. Second, VGVAPGprovides a binding site for elastin-binding protein, a permanentcomponent of mature elastic fibers. Third, VGVAPG provides a bindingsite for elastin and extracellular matrix degradation enzymes such asmatrix metalloproteinases (MMPs), which facilitate the replacement andregeneration of elastic fibers and extracellular matrix proteins.

The mechanism by which the peptide combination of the embodiments worksto stimulate and restore elastin and collagen levels in the skin isdepicted in FIG. 1. The exemplary composition contains a tripeptide anda hexapeptide. The schematic demonstrates the beneficial effects of thetripeptide and hexapeptide, which work synergistically to promote skinregeneration and wound healing through the attraction of healing cells,increased production of elastin and collagen, enhanced fibroblastproliferation, antioxidant behavior (preventing the release of oxidizingiron), and inducing the regeneration of the extracellular matrix. As aresult, the combination of the two peptides exhibits synergistic,superior performance well beyond that expected for either of the twopeptides alone.

FIG. 2 provides a schematic diagram showing a tripeptide's impact onpromoting skin regeneration through increased collagen and elastinsynthesis, blocking ferritin release of oxidized iron, attractinghealing cells such as capillary cells and macrophages, and throughre-establishing new blood flow to the injury site. The tripeptidefunctions as an anti-oxidant, stimulates collage, elastin, andhyaluronic acid. It is formulated to penetrate stratum corneum. In theextracellular matrix (ECM), it is an anti-oxidant, attracts capillariesand macrophages, which facilitates wound healing. In the cell, itdecreases inflammatory cytokines, increases collagen, elastin, dermalstem cell proliferation, and hyaluronic acid.

FIG. 3 provides a schematic diagram showing a hexapeptide's impact onpromoting skin regeneration and wound healing through the induction ofelastin and collagen production, fibroblast proliferation, regenerationof the extracellular matrix, and fibroblast keratinocyte mobility. Thehexapeptide is formulated to penetrate the stratum corneum, and mimicsthe elastin binding sequence, to stimulate elastin. It bindsspecifically to EBP receptors on fibroblasts and keratinocytes. Thebinding initiates intracellular signal transduction.

In topical formulations, the tripeptide is typically present in anamount of from about 50 ppm or less to about 100, 200, 300, 400, or 500ppm or more, e.g., 50 ppm to 150 ppm.

In topical formulations, the hexapeptide is typically present in anamount of from about 50 ppm or less to about 100, 200, 300, 400, or 500ppm or more, e.g., 50 ppm to 150 ppm.

The peptides can advantageously be provided in a base for suitable forcombining with other components of a topical formulation. The base caninclude one or more components such as a thickener/binding agent (e.g.,pentaerythrityl tetraisostearate), an emollient/dispersing agent (e.g.,caprylic/capric triglyceride), a solvent (e.g., propylene carbonate),and/or a rheology modifier/antisettling agent (e.g., disteardimoniumhectorite).

Oleuropein

In some embodiments, polyphenols such as oleuropein may be added to thecompositions. Oleuropein is a polyphenol isolated from olive leaves (seee.g. Omar S H. Oleuropein in olive and its pharmacological effects. SciPharm 2010; 78(2): 133-54; Al-Rimawi F, Yateem H, Afaneh I. Formulationand evaluation of a moisturizing day cream containing olive leavesextract. International Journal of Development Research 2014; 4(10):1996-2000; Kontogianni V G, Charisiadis P, Margianni E, Lamari F N,Gerothanassis I P, Tzakos A G. Olive leaf extracts are a natural sourceof advanced glycation end product inhibitors. Journal of medicinal food2013; 16(9): 817-22). Oleuropein demonstrates major anti-inflammatoryeffects by inhibiting lypoxygenase activity and the production ofleukotriene. More particularly researchers have demonstrated thatoleuropein enhances proteasome activities in vitro more effectively thanother known chemical activators, possibly through conformational changesof the proteasome. In this regard, it decreases reactive oxygen species(ROS), reduces the amount of oxidized proteins through increasedproteasome-mediated degradation through increased proteasome-mediateddegradation and autophagic pathways, and retains proteasome functionduring replicative senescence. Inhibition of AGE formation via blockingsugar attachment to proteins, scavenging the reactive intermediates, orbreakdown of established AGE-induced cross-links constitutes anattractive therapeutic/preventive target. Oleuropein has beendemonstrated to inhibit AGE formation and breakdown AGE products throughits proteasome enhancing function. When oleuropein is employed in atopical formulation, it is preferably present at from about 0.005% byweight or less to about 10.0% by weight or more, typically at from about0.01% by weight to about 5.0% by weight, e.g., at from about 0.05% byweight to about 0.1% by weight. Oleuropein is useful in compositions forpromoting healing. Oleuropein is typically not employed in antiagingcompositions, in that its effects tend to be incompatible withvolumizing, but it can advantageously be employed in formulations forpreconditioning the skin in advance of procedures as described herein(e.g., laser resurfacing, chemical peel, etc.).

Phosphatidyl Serine

In certain embodiments, phospholipids such as phosphatidylserine (PS), ahighly enriched membrane phospholipid component, may be added.Phosphatidylserine has been known to have several physiological roles,such as activating signaling enzymes and antioxidant activity (see e.g.Draelos, Z., Pugliese, P. Glycation and Skin Aging: A Review. Cosmetics& Toiletries Magazine 2011; June 2011: 1-6; Lee, S., Yang, J., Park Y.,et al. Protective effect and mechanism of phosphatidylserine inUVB-induced human dermal fibroblasts. European Journal of Lipid Scienceand Technology 2013; 115(7): 783-90; He, M., Kubo, H., Morimoto, K., etal. Receptor for advanced glycation end products binds tophosphatidylserine and assists in the clearance of apoptotic cells. EMBOreports 2011; 12(4): 358-64). It has been found to decrease MMP-1 in adose dependent manner, to increase procollagen formation and may act asa substrate for AGE targets thus reducing the damage from glycationeffects. Clearance of apoptotic cells is necessary for tissuedevelopment, homeostasis, and resolution of inflammation.Phosphatidylserine provides an “eat me” signal on the cell surface, andphagocytes recognize the signal using specific receptors such as thereceptor of advanced glycation end-products (RAGE). This then binds toPS and assists in the clearance of apoptotic cells and end products ofAGE. When phosphatidylserine is employed in a topical formulation, it ispreferably present at from about 0.005% by weight or less to about 10.0%by weight or more, typically at from about 0.01% by weight to about 5.0%by weight, e.g., at from about 0.05% by weight to about 0.1% by weight.

Phosphatidylserine can advantageously be employed in formulations forpreconditioning the skin in advance of procedures as described herein(e.g., laser resurfacing, chemical peel, etc.).

Carrier Systems

Liquids and gels containing the peptides and other components asdescribed herein can be prepared using techniques as are known in theart of cosmetics manufacture. See, e.g., Handbook of Cosmetic Scienceand Technology, Fourth Edition, edited by André O. Barel, Marc Paye,Howard I. Maibach, CRC Press, 2014, the contents of which is herebyincorporated by reference in its entirety. Various formulations arepossible. As an example, a clear cosmetic gel stick composition caninclude 60 to about 90% of an aliphatic polyhydric alcohol (e.g., a C2-6alcohol containing from 2 to 6 hydroxyl groups); 1-10% of a soap; and1-10% of a water-soluble emollient, e.g., a polyoxyalkylene ether of aC8-22 fatty alcohol, as the main ingredients, in combination with thepeptides of the preferred embodiments. Aqueous extrudable gels are basedon water-oil emulsion technologies. To minimize the amount of waterintroduced into an extrudable gel formula, the concentration of theactive solution is adjusted. Ideally, a high concentration activesolution (45-50%) of the peptides can be employed. Carrier systems forAP solids are typically based on volatile cyclic siloxanes because theyevaporate quickly and do not leave residue on the skin. As analternative to volatile cyclic siloxanes, alternatives can be used,including isohexadecane or C13-15 isoalkane. Solidification systems areemployed to develop solid sticks that do not melt under typical storageor consumer conditions but provide an elegant skin feel and allow foreasy transfer. A combination of cyclopentasiloxane and stearyl alcoholwith varying degrees of additional waxes such as hydrogenated castorwax, hydrogenated vegetable oils and polyethylene, can be employed.

For liquid formulations (e.g., gel or lotion forms), a silicone, e.g., acyclosiloxane or linear silicone (e.g., silicone elastomer), can beemployed as a carrier. One type of suitable carrier is a dimethiconecrosspolymer gel, e.g., dimethicone crosspolymer in cyclopentasiloxane.Other suitable dimethicone crosspolymers include cyclopentasiloxane,dimethicone/vinyldimethicone crosspolymer; dimethicone,dimethicone/vinyl dimethicone crosspolymer; and isodecanedimethicone/vinyl dimethicone crosspolymer.

Typically, the carrier is present in an amount of from about 80 wt. % toabout 95 wt. %, or 82 wt. % to 92 wt. %, e.g., in a topical formulationfor application to skin or mucous membranes.

Penetration Enhancers

Heptyl undecylenate can be employed to enhance penetration of thepeptides, and to provide a silky feel to formulations. Other fatty acidesters can also be employed, e.g., methanoic acid, ethanoic acid,propanoic acid, butanoic acid, isobutyric acid, pentanoic acid, hexanoicacid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid,myristoleic acid, isovaleric acidpalmitoleic acid, sapienic acid, oleicacid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid,α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid,docosahexaenoic acid, caprylic acid, capric acid, lauric acid, palmiticacid, stearic acid, arachidic acid, behenic acid, lignoceric acid,cerotic acid, medium chain fatty acids, e.g., C₆₋₁₂ fatty acids, or thelike. Typical amounts when employed in topical formulations are from 1%by weight to 4% by weight. While heptyl undecylenate can advantageouslybe employed in most formulations, it can be considered an optionalingredient in glider formulations for use in conjunction withmicroneedling, and may be omitted for this use.

Anti-Irritation Agents

Panthenyl triacetate/naringenin are natural plant extracts that reduceredness and water loss through the skin. Typical amounts foranti-irritation agents when employed in topical formulations are from 1%by weight to 4% by weight.

Anti-Inflammatory Agents

Arnica montana extract includes components such as essential oils, fattyacids, thymol, pseudoguaianolide sesquiterpene lactones and flavanoneglycosides. It can exhibit an anti-inflammatory effect. Typical amountsfor anti-inflammatory agents when employed in topical formulations arefrom 1% by weight to 4% by weight.

Antioxidant Agents

Dunaliella salina extract includes components such as beta carotenes. Itcan exhibit an antioxidant effect. Typical amounts for anti-inflammatoryagents when employed in topical formulations are from 0.1% by weight to2% by weight.

Solubility Enhancers

Certain components of the formulation tend to be difficult to solubilizein conventional formulations. For example, phosphatidyl serine andoleuropein are known to exhibit solubility issues. It has been foundthat a siloxane polymer, e.g., caprylyl methicone, is particularlyeffective at solubilizing these two components in anhydrousformulations. For topical compositions containing from about 0.05% byweight to about 0.1% by weight phosphatidyl serine and/or from about0.05% by weight to about 0.1% by weight oleuropein, caprylyl methiconein an amount of from about 0.5% by weight to about 1% by weight ofcaprylyl methicone can solubilize these components in an anhydrousformulation.

Hectorite Clays

Hectorite clays, such as modified hectorite clays, can be employed inconjunction with the peptides to provide impart penetration andadsorption properties to the compositions, and can aid in stabilizingemulsions. Hectorite has the chemical formulaNa_(0.3)(Mg,Li)₃Si₄O₁₀(OH)₂. Other clays, such as bentonite andmagnesium aluminum silicate can also be employed.

Hectorite or other clays can be modified to yield an organic modifiedclay compound. Salts (e.g., quaternary ammonium salts) of fatty acids(e.g., hydrogenated fatty acids) can be reacted with hectorite or otherclays. As provided herein, fatty acids are referred to and describedusing conventional nomenclature as is employed by one of skill in theart. A saturated fatty acid includes no carbon-carbon double bonds. Anunsaturated fatty acid includes at least one carbon-carbon double bond.A monounsaturated fatty acid includes only one carbon-carbon doublebond. A polyunsaturated fatty acid includes two or more carbon-carbondouble bonds. Double bonds in fatty acids are generally cis; however,trans double bonds are also possible. The position of double bonds canbe indicated by Δn, where n indicates the lower numbered carbon of eachpair of double-bonded carbon atoms. A shorthand notation specifyingtotal # carbons:# double bonds, Δ_(double bond positions) can beemployed. For example, 20:4Δ_(5,8,11,14) refers to a fatty acid having20 carbon atoms and four double bonds, with the double bonds situatedbetween the 5 and 6 carbon atom, the 8 and 9 carbon atom, the 11 and 12carbon atom, and the 14 and 15 carbon atom, with carbon atom 1 being thecarbon of the carboxylic acid group. Stearate (octadecanoate) is asaturated fatty acid. Oleate (cis-Δ9-octadecenoate) is a monounsaturatedfatty acid, linolenate (all-cis-Δ9,12,15-octadecatrienoate) is apolyunsaturated fatty acid. Fatty acids suitable for use can comprisefrom 5 to 30 carbon atoms, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbonatoms. The fatty acid can be fully saturated, or can include as manydouble bonds as are feasible for the chain length. Fatty acids suitablefor functionalizing hectorite or other clays include palmitic acid andstearic acid. Dialkyl quaternary cationic modifiers includedipalmoyldimonium chloride and distearyldimonium chloride. Amidoaminequaternary cationic modifiers include palmitamidopropyltrimoniumchloride cetearyl alcohol and palmitamidopropyltrimonium chloride.

Other Excipients and Agents

In some embodiments, the peptides can be in admixture with a suitablecarrier, diluent, or excipient, and can contain auxiliary substancessuch as wetting or emulsifying agents, pH buffering agents, gelling orviscosity enhancing additives, preservatives, scenting agents, colors,and the like, depending upon the route of administration and thepreparation desired. See, e.g., “Remington: The Science and Practice ofPharmacy”, Lippincott Williams & Wilkins; 20th edition (Jun. 1, 2003)and “Remington's Pharmaceutical Sciences,” Mack Pub. Co.; 18th and 19theditions (December 1985, and June 1990, respectively). Such preparationscan include complexing agents, metal ions, polymeric compounds such aspolyacetic acid, polyglycolic acid, hydrogels, dextran, and the like,liposomes, microemulsions, micelles, unilamellar or multilamellarvesicles, erythrocyte ghosts or spheroblasts. Suitable lipids forliposomal formulations include, without limitation, monoglycerides,diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bileacids, and the like. The presence of such additional components caninfluence the physical state, solubility, stability, rate of release,rate of clearance, and penetration of active ingredients.

The compositions for topical administration comprise the peptidecompositions as described herein and a dermatologically acceptablevehicle. The vehicle may be aqueous or nonaqueous. The dermatologicallyacceptable vehicle used in the topical composition may be in the form ofa lotion, a gel, an ointment, a liquid, a cream, or an emulsion. If thevehicle is an emulsion, the emulsion may have a continuous aqueous phaseand a discontinuous nonaqueous or oil phase (oil-in-water emulsion), ora continuous nonaqueous or oil phase and a discontinuous aqueous phase(water-in-oil emulsion). When administered topically in liquid or gelform, a liquid carrier such as water, petroleum, oils of animal or plantorigin such as peanut oil, mineral oil, soybean oil, or sesame oil, orsynthetic oils can be added to the active ingredient(s). Physiologicalsaline solution, dextrose, or other saccharide solution, or glycols suchas ethylene glycol, propylene glycol, or polyethylene glycol are alsosuitable liquid carriers. The pharmaceutical compositions can also be inthe form of oil-in-water emulsions. The oily phase can be a vegetableoil, such as olive or arachis oil, a mineral oil such as liquidparaffin, or a mixture thereof. Suitable emulsifying agents includenaturally-occurring gums such as gum acacia and gum tragacanth,naturally occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan mono-oleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. Theemulsions can also contain coloring and scenting agents.

In certain embodiments, a silicone elastomer (e.g., dimethiconecrosspolymer) is employed to increase delivery and penetration of thepeptides into the skin. An alternative to increasing molecular weight(as with silicone gums) or adding filler (as with silicone compounds) isto partially crosslink siloxane polymers and disperse this material inan appropriate silicone carrier fluid. The resulting dimethiconecrosspolymers (also known as silicone elastomers in the personal careindustry) differ from basic polydimethylsiloxane (PDMS) because of thecross-linking between the linear polymers. These materials can beemployed in peptide formulations, and also offer benefits in scartreatment, periwound protection and enzyme delivery. In skin careapplications, the aesthetics of silicone elastomers (including thosewith functional groups) and their ability to absorb various oils (e.g.,with a dimethicone/vinyl dimethicone crosspolymer such as Dow Corning®9506 Elastomer Powder) are two of the elastomer's desirable properties.Silicone elastomers have a skin feel different from any of the siliconefluids, described as “smooth”, “velvety” and “powdery”. It can bemodified by controlling the amount of liquid phase in the formula, andtherefore the degree of swelling. Due to their film-forming properties,dimethicone crosspolymers can be used as delivery systems for activeingredients such as the peptides described herein, or other formulationcomponents such as oil-soluble vitamins and sunscreens. Sunscreens suchas octyl methoxycinnamate can be more efficiently delivered from aformulation containing a silicone elastomer, producing a higher sunprotection factor (SPF). Silicone elastomer blends can be used toenhance SPF in oil-in-water formulations containing organic sunscreens.For example, in testing conducted regarding SPF, the addition of 4%silicone elastomer blend to a suncare formulation containing organicsunscreens increased the SPF from 5.7 to 18. This property of thesilicone elastomer allows the effectiveness of sunscreen agents in aformulation to be maximized while reducing the amount needed to achievea desired SPF. As a result, formulation costs can be reduced along withpotential irritation caused by sunscreen actives. Accordingly, a higherSPF can be achieved with the same amount of UV absorber, resulting inenhanced performance with no added formulation cost. Silicone elastomerscan be produced from linear silicone polymers by a variety ofcrosslinking reactions, e.g., by a hydrosilylation reaction in which avinyl group reacts with a silicon hydride. The general process involveslinear silicone polymers with reactive sites along the polymer chainreacting with a cross-linker. The dimethicone crosspolymer can beproduced either as a gel made of a suspension of elastomer particlesswollen in a carrier fluid (e.g., a mixture of high molecular weightsilicone elastomer in cyclopentasiloxane such as Dow Corning® 9040Silicone Elastomer Blend), or as a spray-dried powder (adimethicone/vinyl dimethicone crosspolymer such as Dow Corning® 9506Elastomer Powder). The gel form having desirable attributes iscyclomethicone, but low viscosity dimethicones and organic fluids canalso be used. Examples of dimethicone crosspolymers in the suspension orgel form are high molecular weight silicone elastomer (12%) indecamethylcyclopentasiloxane (e.g., Dow Corning® ST-Elastomer 10) and amixture of high molecular weight silicone elastomer incyclopentasiloxane (e.g., Dow Corning® 9040 Silicone Elastomer Blend),which typically have an elastomer content ranging from 10 to 20% byweight.

The pharmaceutical excipients used in the topical preparations of thepeptide compositions may be selected from the group consisting ofsolvents, emollients and/or emulsifiers, oil bases, preservatives,antioxidants, tonicity adjusters, penetration enhancers andsolubilizers, chelating agents, buffering agents, surfactants, one ormore polymers, and combinations thereof.

Suitable solvents for an aqueous or hydrophilic topical formulationinclude water; ethyl alcohol; isopropyl alcohol; mixtures of water andethyl and/or isopropyl alcohols; glycerin; ethylene, propylene orbutylene glycols; DMSO; and mixtures thereof. Suitable solvents forhydrophobic topical formulations include mineral oils, vegetable oils,and silicone oils. If desired, the peptide compositions as describedherein may be dissolved or dispersed in a hydrophobic oil phase, and theoil phase may then be emulsified in an aqueous phase comprising water,alone or in combination with lower alcohols, glycerin, and/or glycols.It is generally preferred to employ anhydrous compositions, as thepresence of water can result in stinging upon administration to skintissues subject to laser treatment, chemical peel, dermabrasion, or thelike. Anhydrous formulations may also act to prevent the development ofwater-based irritant contact dermatitis in damaged or sensitive skin,which may produce rashes and skin irritation that may retard woundhealing and improvement in skin quality. Tsai, T. F., Maibach, H. I. Howirritant is water? An overview. Contact Dermatitis 41(6) (1999): 311-314(describing contact dermatitis caused by water as an irritant). However,in certain embodiments it may be acceptable to provide water basedcompositions, or to permit a limited amount of water to be present. Forexample, water may be present, but at amounts below the threshold atwhich a stinging sensation when applied to damaged skin may result.Osmotic shock or osmotic stress is a sudden change in the soluteconcentration around a cell, causing a rapid change in the movement ofwater across its cell membrane. Under conditions of high concentrationsof either salts, substrates or any solute in the supernatant, water isdrawn out of the cells through osmosis. This also inhibits the transportof substrates and cofactors into the cell thus “shocking” the cell.Alternatively, at low concentrations of solutes, water enters the cellin large amounts, causing it to swell and either burst or undergoapoptosis. Certain of the formulations as described herein can beadvantageously employed where it is desirable to minimize osmotic shock.

Viscosity of the compositions can be maintained at the selected levelusing a pharmaceutically acceptable thickening agent. Suitable viscosityenhancers or thickeners which may be used to prepare a viscous gel orcream with an aqueous base include sodium polyacrylate, xanthan gum,polyvinyl pyrrolidone, acrylic acid polymer, carragenans, hydroxyethylcellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose,propyl cellulose, hydroxypropyl methyl cellulose, polyethoxylatedpolyacrylamides, polyethoxylated acrylates, and polyethoxylated alkanethiols. Methylcellulose is preferred because it is readily andeconomically available and is easy to work with. Other suitablethickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropyl cellulose, carbomer, and the like. Thepreferred concentration of the thickener will depend upon the thickeningagent selected. An amount is preferably used that will achieve theselected viscosity. Viscous compositions are normally prepared fromsolutions by the addition of such thickening agents, or by employing abase that has an acceptable level of viscosity.

Suitable emollients include hydrocarbon oils and waxes such as mineraloil, petrolatum, paraffin, ceresin, ozokerite, microcrystalline wax,polyethylene, squalene, perhydrosqualene, silicone oils, triglycerideesters, acetoglyceride esters, such as acetylated monoglycerides;ethoxylated glycerides, such as ethoxylated glyceryl monostearate; alkylesters of fatty acids or dicarboxylic acids.

Suitable silicone oils for use as emollients include dimethylpolysiloxanes, methyl(phenyl) polysiloxanes, and water-soluble andalcohol-soluble silicone glycol copolymers. Suitable triglyceride estersfor use as emollients include vegetable and animal fats and oilsincluding castor oil, safflower oil, cotton seed oil, corn oil, oliveoil, cod liver oil, almond oil, avocado oil, palm oil, sesame oil, andsoybean oil.

Suitable esters of carboxylic acids or diacids for use as emollientsinclude methyl, isopropyl, and butyl esters of fatty acids. Specificexamples of alkyl esters including hexyl laurate, isohexyl laurate,iso-hexyl palmitate, isopropyl palmitate, decyl oleate, isodecyl oleate,hexadecyl stearate, decyl stearate, isopropyl isostearate, dilauryllactate, myristyl lactate, and cetyl lactate; and alkenyl esters offatty acids such as oleyl myristate, oleyl stearate, and oleyl oleate.Specific examples of alkyl esters of diacids include diisopropyladipate, diisohexyl adipate, bis(hexyldecyl) adipate, and diisopropylsebacate.

Other suitable classes of emollients or emulsifiers which may be used inthe topical formulations include fatty acids, fatty alcohols, fattyalcohol ethers, ethoxylated fatty alcohols, fatty acid esters ofethoxylated fatty alcohols, and waxes.

Specific examples of fatty acids for use as emollients includepelargonic, lauric, myristic, palmitic, stearic, isostearic,hydroxystearic, oleic, linoleic, ricinoleic, arachidic, behenic, anderucic acids. Specific examples of fatty alcohols for use as emollientsinclude lauryl, myristyl, cetyl, hexadecyl, stearyl, isostearyl,hydroxystearyl, oleyl, ricinoleyl, behenyl, and erucyl alcohols, as wellas 2-octyl dodecanol.

Specific examples of waxes suitable for use as emollients includelanolin and derivatives thereof including lanolin oil, lanolin wax,lanolin alcohols, lanolin fatty acids, isopropyl lanolate, ethoxylatedlanolin, ethoxylated lanolin alcohols, ethoxolated cholesterol,propoxylated lanolin alcohols, acetylated lanolin, acetylated lanolinalcohols, lanolin alcohols linoleate, lanolin alcohols recinoleate,acetate of lanolin alcohols recinoleate, acetate of lanolin alcoholsrecinoleate, acetate of ethoxylated alcohols esters, hydrogenolysates oflanolin, hydrogenated lanolin, ethoxylated hydrogenated lanolin,ethoxylated sorbitol lanolin, and liquid and semisolid lanolin. Alsousable as waxes include hydrocarbon waxes, ester waxes, and amide waxes.Useful waxes include wax esters such as beeswax, spermaceti, myristylmyristate and stearyl stearate; beeswax derivatives, e.g.,polyoxyethylene sorbitol beeswax; and vegetable waxes including carnaubaand candelilla waxes.

Polyhydric alcohols and polyether derivatives may be used as solventsand/or surfactants in the topical formulations. Suitable polyhydricalcohols and polyethers include propylene glycol, dipropylene glycol,polypropylene glycols 2000 and 4000, poly(oxyethylene-co-oxypropylene)glycols, glycerol, sorbitol, ethoxylated sorbitol,hydroxypropylsorbitol, polyethylene glycols 200-6000, methoxypolyethylene glycols 350, 550, 750, 2000 and 5000, poly[ethylene oxide]homopolymers (100,000-5,000,000), polyalkylene glycols and derivatives,hexylene glycol, 2-methyl-2,4-pentanediol, 1,3-butylene glycol,1,2,6-hexanetriol, 2-ethyl-1,3-hexanediol, vicinal glycols having 15 to18 carbon atoms, and polyoxypropylene derivatives of trimethylolpropane.

Polyhydric alcohol esters may be used as emulsifiers or emollients.Suitable polyhydric alcohol esters include ethylene glycol mono- anddi-fatty acid esters, diethylene glycol mono- and di-fatty acid esters,polyethylene glycol (200-6000) mono- and di-fatty acid esters, propyleneglycol mono- and di-fatty esters, polypropylene glycol 2000 monooleate,polypropylene glycol 2000 monostearate, ethoxylated propylene glycolmonostearate, glyceryl mono- and di-fatty acid esters, polyglycerolpoly-fatty acid esters, ethoxylated glyceryl monostearate, 1,3-butyleneglycol monostearate, 1,3-butylene glycol distearate, polyoxyethylenepolyol fatty acid ester, sorbitan fatty acid esters, and polyoxyethylenesorbitan fatty acid esters.

Suitable emulsifiers for use in topical formulations include anionic,cationic, nonionic, and zwitterionic surfactants. Preferred ionicemulsifiers include phospholipids, such as lecithin and derivatives.

Lecithin and other phospholipids may be used to prepare liposomescontaining the peptide compositions as described herein. Formation oflipid vesicles occurs when phospholipids such as lecithin are placed inwater and consequently form one bilayer or a series of bilayers, eachseparated by water molecules, once enough energy is supplied. Liposomescan be created by sonicating phospholipids in water. Low shear ratescreate multilamellar liposomes. Continued high-shear sonication tends toform smaller unilamellar liposomes. Hydrophobic chemicals can bedissolved into the phospholipid bilayer membrane. The lipid bilayers ofthe liposomes deliver the peptide compositions as described herein.

The topical formulation may contain micelles, or an aggregate ofsurfactant molecules dispersed in an aqueous solution. Micelles may beprepared by dispersing an oil solvent in an aqueous solution comprisinga surfactant, where the surfactant concentration exceeds the criticalmicelle concentration. The resulting formulation contains micelles,i.e., spherical oil droplets surrounded by a membrane of polarsurfactant molecules, dispersed in the aqueous solvent.

Sterols including, for example, cholesterol and cholesterol fatty acidesters; amides such as fatty acid amides, ethoxylated fatty acid amides,and fatty acid alkanolamides may also be used as emollients and/orpenetration enhancers.

A pharmaceutically acceptable preservative can be employed to increasethe shelf life of the composition. Other suitable preservatives and/orantioxidants for use in topical formulations include benzalkoniumchloride, benzyl alcohol, phenol, urea, parabens, butylatedhydroxytoluene (BHT), butylated hydroxyanisole (BHA), tocopherol,thimerosal, chlorobutanol, or the like, and mixtures thereof, can beemployed. If a preservative, such as an antioxidant, is employed, theconcentration is typically from about 0.02% to about 2% based on thetotal weight of the composition, although larger or smaller amounts canbe desirable depending upon the agent selected. Reducing agents, asdescribed herein, can be advantageously used to maintain good shelf lifeof the formulation. It is generally observed that the anhydrousformulations of the embodiments exhibit satisfactory stability, suchthat a preservative can be omitted from the formulation.

Suitable chelating agents for use in topical formulations includeethylene diamine tetraacetic acid, alkali metal salts thereof alkalineearth metal salts thereof, ammonium salts thereof, and tetraalkylammonium salts thereof.

The carrier preferably has a pH of between about 4.0 and 10.0, morepreferably between about 6.8 and about 7.8. The pH may be controlledusing buffer solutions or other pH modifying agents. Suitable pHmodifying agents include phosphoric acid and/or phosphate salts, citricacid and/or citrate salts, hydroxide salts (i.e., calcium hydroxide,sodium hydroxide, potassium hydroxide) and amines, such astriethanolamine. Suitable buffer solutions include a buffer comprising asolution of monopotassium phosphate and dipotassium phosphate,maintaining a pH of between 5.8 and 8; and a buffer comprising asolution of monosodium phosphate and disodium phosphate, maintaining apH of between 6 and 7.5. Other buffers include citric acid/sodiumcitrate, and dibasic sodium phosphate/citric acid. The peptidecompositions of the embodiments are preferably isotonic with the bloodor other body fluid of the recipient. The isotonicity of thecompositions can be attained using sodium tartrate, propylene glycol orother inorganic or organic solutes. Sodium chloride is particularlypreferred. Buffering agents can be employed, such as acetic acid andsalts, citric acid and salts, boric acid and salts, and phosphoric acidand salts. It can be desirable to include a reducing agent in theformulation, such as vitamin C, vitamin E, or other reducing agents asare known in the pharmaceutical arts.

Surfactants can also be employed as excipients, for example, anionicdetergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinateand dioctyl sodium sulfonate, cationic such as benzalkonium chloride orbenzethonium chloride, or nonionic detergents such as polyoxyethylenehydrogenated castor oil, glycerol monostearate, polysorbates, sucrosefatty acid ester, methyl cellulose, or carboxymethyl cellulose.

When the peptide formulations of the embodiments are administered bysubcutaneous injection, it is preferably in the form of a pyrogen-free,parenterally acceptable aqueous solution or oleaginous suspension,emulsion or solution. Suspensions can be formulated according to methodswell known in the art using suitable dispersing or wetting agents andsuspending agents. The preparation of acceptable aqueous or nonaqueoussolutions with suitable properties, e.g., pH, isotonicity, stability,and the like, is within the skill in the art. For example, an isotonicvehicle such as 1,3-butanediol, water, isotonic sodium chloridesolution, Ringer's solution, dextrose solution, dextrose and sodiumchloride solution, lactated Ringer's solution, or other vehicles as areknown in the art can be employed, or a fixed oil can be employedconventionally as a solvent or suspending medium, e.g., synthetic monoor diglycerides, fatty acids, or the like. The peptide formulations canalso contain stabilizers, preservatives, buffers, antioxidants, or otheradditives known to those of skill in the art.

In certain embodiments, it can be advantageous to include additionalagents having pharmacological activity. Anti-infective agents include,but are not limited to, anthelmintic (mebendazole), antibioticsincluding aminoglycosides (gentamicin, neomycin, tobramycin), antifungalantibiotics (amphotericin b, fluconazole, griseofulvin, itraconazole,ketoconazole, nystatin, micatin, tolnaftate), cephalosporins (cefaclor,cefazolin, cefotaxime, ceftazidime, ceftriaxone, cefuroxime,cephalexin), beta-lactam antibiotics (cefotetan, meropenem),chloramphenicol, macrolides (azithromycin, clarithromycin,erythromycin), penicillins (penicillin G sodium salt, amoxicillin,ampicillin, dicloxacillin, nafcillin, piperacillin, ticarcillin),tetracyclines (doxycycline, minocycline, tetracycline), bacitracin,clindamycin, colistimethate sodium, polymyxin b sulfate, vancomycin,antivirals including acyclovir, amantadine, didanosine, efavirenz,foscarnet, ganciclovir, indinavir, lamivudine, nelfinavir, ritonavir,saquinavir, stavudine, valacyclovir, valganciclovir, zidovudine,quinolones (ciprofloxacin, levofloxacin), sulfonamides (sulfadiazine,sulfisoxazole), sulfones (dapsone), furazolidone, metronidazole,pentamidine, sulfanilamidum crystallinum, gatifloxacin, andsulfamethoxazole/trimethoprim. Anesthetics can include, but are notlimited to, ethanol, bupivacaine, chloroprocaine, levobupivacaine,lidocaine, mepivacaine, procaine, ropivacaine, tetracaine, desflurane,isoflurane, ketamine, propofol, sevoflurane, codeine, fentanyl,hydromorphone, marcaine, meperidine, methadone, morphine, oxycodone,remifentanil, sufentanil, butorphanol, nalbuphine, tramadol, benzocaine,dibucaine, ethyl chloride, xylocaine, and phenazopyridine.Anti-inflammatory agents include but are not limited to, nonsteroidalanti-inflammatory drugs (NSAIDs) such as aspirin, celecoxib, cholinemagnesium trisalicylate, diclofenac potassium, diclofenac sodium,diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin,ketoprofen, ketorolac, melenamic acid, nabumetone, naproxen, naproxensodium, oxaprozin, piroxicam, rofecoxib, salsalate, sulindac, andtolmetin; and corticosteroids such as cortisone, hydrocortisone,methylprednisolone, prednisone, prednisolone, betamethesone,beclomethasone dipropionate, budesonide, dexamethasone sodium phosphate,flunisolide, fluticasone propionate, triamcinolone acetonide,betamethasone, fluocinonide, betamethasone dipropionate, betamethasonevalerate, desonide, desoximetasone, fluocinolone, triamcinolone,clobetasol propionate, and dexamethasone.

In certain embodiments, the addition of emollients, emulsionstabilizers, moisturizers, excipients, and other compounds may bemodified to enhance the sensory properties of the topical compositions,including but not limited to: skin feel (silkiness, lightness,creaminess, etc.), absorbency (required time at which product loses wetfeel and is no longer perceived on skin), consistency, firmness,spreadability (e.g. viscosity, flow onset, shear rates), stickiness,integrity of shape, glossiness, hydrophilicity or hydrophobicity, andothers. Preferably, compositions will have high spreadability and lowviscosity properties. Compositions with such properties have beendemonstrated to have an enhanced “silky” or “light” skin feel rating(see e.g. Bekker, M. Webber, G., Louw, N. Relating rheologicalmeasurements to primary and secondary skin feeling when mineral-basedand Fischer-Tropsch wax-based cosmetic emulsions and jellies are appliedto the skin, International Journal of Cosmetic Science 2013, 35(4), pp.354-61).

The compositions can be advantageously employed as a carrier forplatelet-rich plasma (PRP), which is blood plasma that has been enrichedwith platelets. As a concentrated source of autologous platelets, PRPcontains several different growth factors and other cytokines that canstimulate healing of soft tissue. Platelet rich plasma therapy utilizesgrowth factors present in alpha granules of platelets in an autologousmanner, e.g., for treatment of androgenetic alopecia, in wound healing,in facial rejuvenation, or the like For preparation of PRP, variousprotocols are used and no standard protocol exists but main principlesessentially involve concentrating platelets in a concentration of 3-5times the physiological value and then injecting this concentratedplasma in the tissue where healing or effect is desired. Suchformulations can be particularly advantageous in vaginal rejuvenationapplications.

Stability Testing

Stability testing of the topical formulations can be conducted asfollows.

High temperature testing is now commonly used as a predictor oflong-term stability. High temperature testing can be conducted at 37° C.(98 F) and 45° C. (113° F.). If a product is stored at 45° C. for threemonths (and exhibits acceptable stability) then it should be stable atroom temperature for two years. Of course, the product must be stored at25° C. (77° F.) for a period of one year. A good control temperature is4° C. (39° F.) where most products will exhibit excellent stability. Theproduct should also be subjected to −10° C. (14° F.) for three months.

The product should pass three cycles of temperature testing from −10° C.(14° F.) to 25° C. (77° F.). The product is placed at −10° C. for 24hours and place it at room temperature (25° C.) for 24 hours. Thiscompletes one cycle. If the product passes three cycles then you canhave a good degree of confidence in the stability of the product. Aneven more rigorous test is a −10° C. to 45° C. five-cycle test. Thisputs emulsions under a tremendous stress and, if it passes the test,indicates that you have a highly stable product.

The dispersed phase (of an oil-in-water emulsion) has a tendency toseparate and rise to the top of the emulsion forming a layer of oildroplets. This phenomenon is called creaming. Creaming is one of thefirst signs of impending emulsion instability. A test method to predictcreaming is centrifugation. Heat the emulsion to 50° C. (122° F.) andcentrifuge it for thirty minutes at 3000 rpm. Then inspect the resultantproduct for signs of creaming.

Both formulas and packaging can be sensitive to the UV radiation. Theproduct is placed in glass and the actual package in a light box thathas a broad-spectrum output. Another glass jar completely covered withaluminum foil serves as a control. Discoloration of the product may beobserved.

For all the above mentioned tests the color, odor/fragrance, viscosity,pH value, and, if available, particle size uniformity and/or particleagglomeration under the microscope can be observed.

Kits for Non-Invasive Use and Use with Invasive Procedures

Some embodiments of the methods and compositions provided herein includekits comprising peptides provided herein. In some embodiments, kits canbe provided to an administering physician, other health careprofessional, a patient, or a caregiver. In some embodiments, a kitcomprises a container which contains the peptide compositions in asuitable topical formulation, and instructions for administering thepeptide composition to a subject. The kit can optionally also containone or more additional therapeutic or other agents. For example, a kitcontaining a peptide composition in topical form can be provided alongwith other skin care agents, such as, cleansers, occlusive moisturizers,penetrating moisturizers, sunscreens, sunblocks, and the like. The kitmay contain the peptide composition in bulk form, or can containseparate doses of the peptide composition for serial or sequentialadministration. The kit can optionally contain one or more diagnostictools, administration tools, and/or instructions for use. The kit cancontain suitable delivery devices, such as, syringes, pump dispensers,single dose packets, and the like, along with instructions foradministering the peptide compositions and any other therapeutic orbeneficial agents. The kit can optionally contain instructions forstorage, reconstitution (if applicable), and administration of any orall therapeutic or beneficial agents included. The kits can include aplurality of containers reflecting the number of administrations to begiven to a subject, or the different products to be administered to thesubject.

The topical peptide formulation, in addition to the tripeptide andhexapeptide as described herein such as, as palmitoyl hexapeptide-12 andpalmitoyl tripeptide-1, can contain other ingredients. For example,other ingredients can include cyclopentasiloxane, dimethiconecrosspolymer, pentaerythrityl tetraisostearate, heptyl undecylenate,glycine soja (soybean) oil, panthenyl triacetate, narigenin, arnicamontana extract, dunaliella salina extract, disteardimonium hectorite,tocopherol, squalane, caprylic/capric triglyceride, stearalkoniumhectorite, and propylene carbonate. In some embodiments, the formulationis configured to support the skin before, during and after cosmeticprocedures, and also works with the skin's own natural regeneratingprocess and assists in improving the skin's appearance. The topicalpeptide formulation can be applied immediately post-procedure for fasterrecovery, or generally for healthier looking skin. The peptideformulation can increase natural levels of elastin in the skin, improvesthe quality of existing elastin, stimulates increase in collagenproduction, and exhibits high antioxidant activity to reduceinflammation, redness and irritation. The topical peptide formulation issuitable for all skin types and post-procedure skin. The topicalformulations can be provided to the patient in bulk form, to permit asuitable amount of the peptides to be self-administered by the patient.For example, the patient can apply an amount of the formulationsufficient to provide an even coating over the affected area or asotherwise instructed by the physician. In certain embodiments it candesirable to incorporate additional therapeutic or active agents intothe topical formulation. Alternatively, adjunct therapies or agents canbe administered separately. For example, a cleanser, a sunblock, asunscreen, a penetrating moisturizer, and/or an occlusive moisturizercan be provided for administration before or after the topicalcomposition of the embodiments.

The topical peptide compositions can be used in conjunction with agentle cleanser. The gentle, self-foaming cleanser removes impuritiesfrom the skin without drying or irritating the skin. The gentle cleansercan contain a blend of moisturizers and vitamins designed to clean,soothe and soften the skin without upsetting the skin's delicatemoisture balance. In use, the cleanser softens, soothes and moisturizesskin, thoroughly removes environmental pollutants and make-up, issulfate-free, and is suitable for use post-procedure on sensitive skin.In one embodiment, the gentle cleanser comprises water, sodium C14-16olefin sulfonate, cocamidopropyl betaine, acrylates copolymer,PEG/PPG-8/3 diisostearate, avena satvia (oat) kernel extract, panthenol,glycerin, beta glucan, bisabolol, lavandula angustifolia (lavender) oil,citrus aurantium dulcis (orange) peel oil, titanium dioxide, tin oxide,synthetic fluorphlogopite, disodium EDTA, phenoxyethanol,ethylhexylglycerin, citric acid, and sodium hydroxide.

The topical peptide compositions can be used in conjunction with anocclusive moisturizer. The occlusive moisturizer is an ointmentformulated to moisturize the skin and work with the body's own naturalrejuvenating processes immediately following cosmetic procedures. Itprotects and enhances post-procedure outcomes for skin. The occlusivemoisturizer softens, soothes and moisturizes skin, supports renewal ofpost-procedure skin, helps restore the skin's moisture balance, hydratesdry and compromised skin, and is suitable for post-procedure skin andextremely dry skin. In one embodiment, the occlusive moisturizercomprises petrolatum, microcrystalline wax, physalis angulata extract,caprylic/capric triglyceride, butyrospermum parki (shea butter) extract,bisabolol, and tocopherol.

The topical peptide compositions can be used in conjunction with apenetrating moisturizer. The moisturizer is a ceramide rich, hydratingmoisturizer formulated to help rebuild the skin's natural barrierfunction, which can be compromised following cosmetic procedures. Themoisturizer contains moisture building ingredients, antioxidants, andsoothing phyto-nutrients that help promote and maintain the barrierfunction of the skin. The moisturizer helps to restore moisture balance,soften, soothe and hydrate skin, inhibit free radicals, and replenishand brighten dull, dry skin. The moisturizer is suitable for all skintypes and post-procedure skin. In on embodiment, the penetratingmoisturizer comprises water, caprylic/capric triglyceride, cetylethylhexanoate, cetaryl alcohol, squalane, niacinamide, dimethicone,cetearyl glucoside, glyceryl stearate, PEG-100 stearate, propanediol,ceramide 3, phytosterols, butyrospermum parklii (shea butter) extract,butyrospermum parklii (shea butter), olea europeaea (olive) fruit oil,sodium hyaluronate, beta-glucan, hydrolyzed pea protein, dunaliellasalina extract, xylitylglucoside, anhydroxylitol, xylitol, glycerin,lecithin, caprylyl glycol, caprylhydroxamic acid, xanthan gum, disodiumEDTA, ethylhexylglycerin, and phenoxyethanol.

The topical peptide compositions can be used in conjunction with asunscreen. The sunscreen can be of a suitable SPF, such as SPF 10+, SPF15+, SPF 20+, SPF 30+, SPF 40+, SPF 50+, or higher. The sunscreen can bea broad spectrum and water resistant, such as 80 minutes or more)sunscreen. The sunscreen can include antioxidants, hydrators and skinsoothing phytonutrients. The sunscreen can be used daily and immediatelyfollowing facial treatments. The sunscreen provides broad spectrumUVA/UVB sun protection, moisturizing UV protection for the face, isnon-comedogenic, is suitable for use post-procedure, is fragrance-freeand paraben-free. Active ingredients of the sunscreen can include zincoxide (e.g., 10% by weight) and octinoxate (e.g., 7.5% by weight). Inone embodiment, the sunscreen comprises water, ethylhexyl palmitate,cyclomethicone, dimethicone, laurylmethicone copolyol, butylene glycol,tocopherol acetate, sodium chloride, aleurites moluccana (kukui nut)seed oil, emelia sinensis leaf (green tea) extract, cucmis staivus(cucumber) fruit extract, aloe barbadensis (aloe vera) leaf extract,tetrahexyldecyl ascorbate, allantoin, sodium hyaluronate, disodium EDTA,methylisothiazolinone, ethylhexlglycerin, and fragrance.

In one embodiment, a kit is provided for use in connection with aninvasive skin procedure, as described herein. The kit, termed “aninvasive kit”, includes a topical peptide composition, an occlusivemoisturizer, a gentle cleanser, a penetrating moisturizer, and a broadspectrum SPF 30+ sunscreen.

In another embodiment, a kit is provided for use in connection withimproving skin health but not in connection with an invasive skinprocedure. The kit, termed “a noninvasive kit”, includes a topicalpeptide composition, a gentle cleanser, a penetrating moisturizer, and abroad spectrum SPF 30+ sunscreen.

The various examples of creams, ointments, lotions, solutions, gels,sprays and patches may incorporate the peptide compositions as describedherein as the active ingredient, in combination with penetrationenhancing agents and other active agents acting synergistically on theskin for the promotion of wound healing or wound closure or thetreatment of chronic cutaneous wound.

Oral Supplements

While topical administration of the peptides of disclosed herein canadvantageously be employed, in certain embodiments systemicadministration can be desirable. In such embodiments, the peptides areformulated into a composition suitable for oral administration, butother routes of administration are also contemplated.

The peptide compositions described herein can be administered bythemselves to a subject, or in compositions where they are mixed withother active agents, as in combination therapy, or with carriers,diluents, excipients or combinations thereof. Formulation is dependentupon the route of administration chosen. Techniques for formulation andadministration of the compounds described herein are known to thoseskilled in the art (see, e.g., “Remington: The Science and Practice ofPharmacy”, Lippincott Williams & Wilkins; 20th edition (Jun. 1, 2003)and “Remington's Pharmaceutical Sciences,” Mack Pub. Co.; 18th and 19theditions (December 1985, and June 1990, respectively).

The peptide combinations disclosed herein may be manufactured intoadministrable forms by a process that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, tableting, or extractingprocesses.

Multiple techniques of administering a compound exist in the artincluding, but not limited to, oral, rectal, topical, aerosol, injectionand parenteral delivery, including intramuscular, subcutaneous,intravenous, intramedullary injections, intrathecal, directintraventricular, intraperitoneal, intranasal and intraocularinjections. Contemplated herein is any combination of the forgoing, orother methods as would be known to one of ordinary skill in the art(see, e.g., “Remington: The Science and Practice of Pharmacy”,Lippincott Williams & Wilkins; 20th edition (Jun. 1, 2003) and“Remington's Pharmaceutical Sciences,” Mack Pub. Co.; 18th and 19theditions (December 1985, and June 1990, respectively).

In practice, the peptides may be combined as the active ingredient inintimate admixture with a pharmaceutical carrier according toconventional pharmaceutical compounding techniques. The ratio of thepeptides to each other is maintained as in the topical formulation,however, excipients are preferably minimized so as to ensureadministration of an appropriate amount of peptides in a compact format.In its simplest form, the peptides can be added directly to, e.g., agelatin capsule or a softgel capsule for consumption by the patient. Inother embodiments, carriers can be employed. The carrier can take a widevariety of forms depending on the form of preparation desired foradministration. Thus, the peptide compositions provided herein can bepresented as discrete units suitable for oral administration such ascapsules, cachets or tablets each containing a predetermined amount ofthe active ingredient. Further, the peptide compositions can bepresented as an oil, a powder, as granules, as a solution, as asuspension in an aqueous liquid, as a non-aqueous liquid, as anoil-in-water emulsion, or as a water-in-oil liquid emulsion, similar tothe topical formulations described elsewhere herein, but usingcomponents suitable for human consumption. In addition to the commondosage forms set out above, the peptide compositions provided herein canalso be administered by controlled release and/or delivery devices. Thepeptide compositions can be prepared by any of the methods of pharmacy.In general, such methods include a step of bringing into association theactive ingredient with the carrier that constitutes one or morenecessary ingredients. In general, the peptide compositions are preparedby uniformly and intimately admixing the peptide ingredients with liquidcarriers or finely divided solid carriers or both. The product can thenbe conveniently shaped into the desired presentation.

A peptide formulation may also be administered in a local rather thansystemic manner, for example, via injection of the peptide compositiondirectly into a target area, e.g., in a depot or sustained releaseformulation. Furthermore, a targeted drug delivery system for thepeptide may be used, for example, in a liposome coated with a tissuespecific antibody.

The peptide compositions may contain the peptides in an amount effectivefor the desired therapeutic effect. In some embodiments, the peptidecompositions are in a unit dosage form and comprise from about 0.1 mg orless to about 5000 mg or more of peptides per unit dosage form. Infurther embodiments, the peptide compositions comprise from about 1 toabout 500 mg per unit dosage form or from about 500 to 5000 mg per unitdosage form of peptides. Such dosage forms may be solid, semisolid,liquid, an emulsion, or adapted for delivery via aerosol or the like.

The carrier employed can be, for example, a solid, liquid, or gas.Examples of solid carriers include lactose, terra alba, sucrose, talc,gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid.Examples of liquid carriers are sugar syrup, peanut oil, olive oil,lower alcohols, and water. Examples of gaseous carriers include carbondioxide and nitrogen.

Peptide compositions provided herein can be prepared as solutions orsuspensions of the peptides in water or nonaqueous liquids. A suitablesurfactant can be included such as, for example, hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof in oils. Further, a preservative can beincluded to, for example, prevent the detrimental growth ofmicroorganisms.

Peptide compositions provided herein suitable for injectable use includesterile aqueous solutions or dispersions. Furthermore, the peptidecompositions can be in the form of sterile powders for theextemporaneous preparation of such sterile injectable solutions ordispersions. The peptide compositions must be stable under theconditions of manufacture and storage; thus, preferably should bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol and liquid polyethylene glycol), vegetable oils, andsuitable mixtures thereof.

In addition to the aforementioned carrier ingredients, the peptideformulations described above can include, as appropriate, one or moreadditional carrier ingredients such as diluents, buffers, flavoringagents, binders, surface-active agents, thickeners, lubricants,preservatives (including anti-oxidants) and the like. Furthermore, otheradjuvants can be included to render the formulation isotonic with theblood or other bodily fluids of the intended recipient. Peptidecompositions can also be prepared in powder or liquid concentrate formfor dilution.

Contemplated herein are peptide compositions including the peptides asdescribed herein in combination with at least one additional activeagent. The peptides and the at least one additional active agent(s) maybe present in a single formulation or in multiple formulations providedtogether, or may be unformulated. In some embodiments, the peptides canbe administered with one or more additional agents together in a singlecomposition. For example, the peptides can be administered in onecomposition, and at least one of the additional agents can beadministered in a second composition. In a further embodiment, thepeptide and the at least one additional active agent(s) are co-packagedin a kit. For example, a drug manufacturer, a drug reseller, aphysician, a compounding shop, or a pharmacist can provide a kitcomprising the peptide in combination with another product or componentfor delivery to a patient. Such additional components can includeanti-infective agents, anti-inflammatory agents, anesthetics, or thelike.

Some embodiments described herein relate to oral compositions ofpeptides, which can include a therapeutically effective amount of thepeptides described herein and a pharmaceutically acceptable carrier,diluent, excipient or combination thereof. The peptide composition caninclude the peptides in an amount for example, >1%, ≥2%, ≥3%, ≥4%, ≥5%,≥6%, ≥7%, ≥8%, ≥9%, ≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%,≥90%, ≥95%, or ≥98% of the composition. As described elsewhere herein,the first peptide and second peptide are present in the composition at aweight ratio of 1 part of the first peptide (or first peptides) to about1 to 2 parts of the second peptide (or second peptides).

EXAMPLES Example 1A: Peptide Formulations

Several peptide-containing topical formulations were prepared comprisinga first peptide and a second peptide in combination with excipients. Theformulations so prepared were evaluated for suitability for use as atopical formulation, including skin feel and stability. The formulationswere prepared as in the following tables.

Formula 1A Ingredient % (wt.) Isododecane (and) DimethiconeCrosspolymer-3 95.468 Physalis Angulata Extract 1.000 Arnica MontanaExtract 2.000 Centella Asiatica Extract 0.250 Squalane 1.000 GrowthHormone Releasing Peptide-6 0.032 1,3 Propanediol 0.250

Formula 2A Ingredient % (wt.) Isododecane (and) DimethiconeCrosspolymer-3 92.718 Panthenyl Triacetate/Naringenin 2.000 ArnicaMontana Extract 2.000 Dunaliella Salina Extract 1.000 Squalane 2.000Growth Hormone Releasing Peptide-2 0.032 1,3 Propanediol 0.250

Formula 3A Ingredient % (wt.) Cyclopentasiloxane, Polysilicone-11 92.718Panthenyl Triacetate/Naringenin 2.000 Arnica Montana Extract 2.000Dunaliella Salina Extract 1.000 Squalane 2.000 Growth Hormone ReleasingPeptide-2 0.032 1,3 Propanediol 0.250

Formula 4A Ingredient % (wt.) Isododecane (and) DimethiconeCrosspolymer-3 87.500 Panthenyl Triacetate/Naringenin 2.000 ArnicaMontana Extract 2.000 Dunaliella Salina Extract 1.000 Squalane 2.000Carrier: 5.500  Pentaerythrityl tetraisostearate  Caprylic/caprictriglyceride  Propylene carbonate  Stearalkonium hectorite Palmitoylhexapeptide-12 (@ 100 ppm in carrier)

Formula 5A Ingredient % (wt.) Isododecane (and) DimethiconeCrosspolymer-3 95.468 Physalis Angulata Extract 1.000 Arnica MontanaExtract 2.000 Centella Asiatica Extract 0.250 Squalane 1.000 GrowthHormone Releasing Peptide-6 0.032 1,3 Propanediol 0.250

Formula 6A Ingredient % (wt.) Isododecane (and) DimethiconeCrosspolymer-3 92.718 Panthenyl Triacetate/Naringenin 2.000 ArnicaMontana Extract 2.000 Dunaliella Salina Extract 1.00 Squalane 2.000Growth Hormone Releasing Peptide-6 0.032 1,3 Propanediol 0.250

Formula 7A Ingredient % (wt.) Cyclopentasiloxane, Polysilicone-11 92.718Panthenyl Triacetate/Naringenin 2.000 Arnica Montana Extract 2.000Dunaliella Salina Extract 1.00 Squalane 2.000 Growth Hormone ReleasingPeptide-6 0.032 1,3 Propanediol 0.250

Formula 8A Ingredient % (wt.) Isododecane (and) DimethiconeCrosspolymer-3 87.500 Panthenyl Triacetate/Naringenin 2.000 ArnicaMontana Extract 2.000 Dunaliella Salina Extract 1.000 Squalane 2.000Carrier: 5.500  Pentaerythrityl tetraisostearate  Caprylic/caprictriglyceride  Propylene carbonate  Stearalkonium hectorite Palmitoylhexapeptide-12* *Present in formulation at 0.055 wt. %; present incarrier at 100 ppm.

Formula 9A Ingredient % (wt.) Isododecane (and) DimethiconeCrosspolymer-3 87.500 Panthenyl Triacetate/Naringenin 2.000 ArnicaMontana Extract 2.000 Dunaliella Salina Extract 1.000 Squalane 2.000Carrier: 5.500  Pentaerythrityl tetraisostearate  Caprylic/caprictriglyceride  Propylene carbonate  Stearalkonium hectorite Palmitoylhexapeptide-12* *Present in formulation at 0.055 wt. %; present incarrier at 100 ppm.

Formula 10A Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 84.500 Heptyl Undecylenate 5.000 PanthenylTriacetate/Naringenin 2.000 Arnica Montana Extract 2.000 DunaliellaSalina Extract 1.000 Carrier: 5.500  Pentaerythrityl tetraisostearate Caprylic/capric triglyceride  Propylene carbonate  Stearalkoniumhectorite Palmitoyl hexapeptide-12* *Present in formulation at 0.055 wt.%; present in carrier at 100 ppm.

Formula 11A Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 84.500 Heptyl Undecylenate 5.000 PanthenylTriacetate/Naringenin 2.000 Arnica Montana Extract 2.000 DunaliellaSalina Extract 1.000 Carrier: 5.500  Pentaerythrityl tetraisostearate Caprylic/capric triglyceride  Propylene carbonate  Stearalkoniumhectorite Palmitoyl hexapeptide-12* *Present in formulation at 0.055 wt.%; present in carrier at 100 ppm.

Formula 12A Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 83.500 Heptyl Undecylenate 3.000 PanthenylTriacetate/Naringenin 2.000 Arnica Montana Extract 2.000 DunaliellaSalina Extract 1.000 Squalane 2.000 Carrier: 5.500   Pentaerythrityltetraisostearate   Caprylic/capric triglyceride   Propylene carbonate  Stearalkonium hectorite Palmitoyl hexapeptide-12* Carrier: 1.000  Pentaerythrityl tetraisostearate   Caprylic/capric triglyceride  Propylene carbonate   Disteardimonium hectorite Palmitoyltripeptide-1** *Present in formulation at 0.055 wt. %; present incarrier at 100 ppm. **Present in formulation at 0.01 wt. %; present incarrier at 100 ppm.

Formula 13A Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 83.500 Heptyl Undecylenate 5.000 PanthenylTriacetate/Naringenin 2.000 Arnica Montana Extract 2.000 DunaliellaSalina Extract 1.000 Carrier: 5.500   Pentaerythrityl tetraisostearate  Caprylic/capric triglyceride   Propylene carbonate   Stearalkoniumhectorite Palmitoyl hexapeptide-12* Carrier: 1.000   Pentaerythrityltetraisostearate   Caprylic/capric triglyceride   Propylene carbonate  Disteardimonium hectorite Palmitoyl tripeptide-1** *Present informulation at 0.055 wt. %; present in carrier at 100 ppm. **Present informulation at 0.01 wt. %; present in carrier at 100 ppm.

Formula 14A Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 83.500 Heptyl Undecylenate 5.000 PanthenylTriacetate/Naringenin 2.000 Arnica Montana Extract 2.000 DunaliellaSalina Extract 1.000 Carrier: 5.500   Pentaerythrityl tetraisostearate  Caprylic/capric triglyceride   Propylene carbonate   Stearalkoniumhectorite Palmitoyl hexapeptide-12* Carrier: 1.000   Pentaerythrityltetraisostearate   Caprylic/capric triglyceride   Propylene carbonate  Disteardimonium hectorite Palmitoyl tripeptide-1** *Present informulation at 0.055 wt. %; present in carrier at 100 ppm. **Present informulation at 0.01 wt. %; present in carrier at 100 ppm.

Formula 15A Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 83.400 Heptyl Undecylenate 2.500 PanthenylTriacetate/Naringenin 2.000 Arnica Montana Extract 2.000 DunaliellaSalina Extract 1.000 Carrier: 5.500   Pentaerythrityl tetraisostearate  Caprylic/capric triglyceride   Propylene carbonate   Stearalkoniumhectorite Palmitoyl hexapeptide-12* Carrier 3.000   Pentaerythrityltetraisostearate   Caprylic/capric triglyceride   Propylene carbonate  Disteardimonium hectorite Palmitoyl tripeptide-1** Caprylyl methicone0.500 Phospatidyl serine/lecithin 0.050 Oleuropein 0.050 *Present informulation at 0.055 wt. %; present in carrier at 100 ppm. **Present informulation at 0.03 wt. %; present in carrier at 100 ppm.

Formula 16A Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 85.900 Heptyl Undecylenate 2.500 PanthenylTriacetate/Naringenin 2.000 Arnica Montana Extract 2.000 DunaliellaSalina Extract 1.000 Carrier: 3.000   Pentaerythrityl tetraisostearate  Caprylic/capric triglyceride   Propylene carbonate   Stearalkoniumhectorite Palmitoyl hexapeptide-12 Carrier: 3.000   Pentaerythrityltetraisostearate   Caprylic/capric triglyceride   Propylene carbonate  Disteardimonium hectorite Palmitoyl tripeptide-1 Caprylyl methicone0.500 Phospatidyl serine/lecithin 0.050 Oleuropein 0.050 *Present informulation at 0.03 wt. %; present in carrier at 100 ppm. **Present informulation at 0.03 wt. %; present in carrier at 100 ppm.

Example 1B: Peptide Formulations

A series of formulations were prepared to assess the impact of variouscomponents on viscosity and stability. As discussed above,phosphatidylserine and oleuropein are difficult to solubilize.

The starting formulation, Formula 1B had a viscosity of approximately5200 cPs. Formula 2B had a viscosity of approximately 6100 cPs. Formula3B had a viscosity of approximately 7000 cPs. Formulation 2B andFormulation 3B each employed a different base, with thecyclopentysiloxane providing a higher viscosity. The results of thiscomparative test demonstrated that cyclopentysiloxane was able toprovide a more desirable level of viscosity in formulation than theisododecane, dimethicone crosspolymer-3.

Formula 4B had a viscosity of approximately 8000 cPs. Formula 5B, whichincluded cyclopentasiloxane as a base, exhibited a viscosity ofapproximately 9500 cPs. Formula 6B, using cyclopentasiloxane,dimethicone crosspolymer (DC9045) and heptyl undecylenate, exhibited aviscosity of approximately 28000 cPs. The results of this comparativetest demonstrated that cyclopentysiloxane was able to provide a moredesirable level of viscosity in formulation than the isododecane,dimethicone crosspolymer-3. The tests further demonstrated that thecombination of cyclopentasiloxane, dimethicone crosspolymer and heptylundecylenate exhibited acceptable levels of viscosity.

Formula 7B was similar to Formula 6B but also including palmitoyltripeptide at 1%, exhibited a viscosity of approximately 16000 cPs—lowerthan that of Formula 6B, but still acceptably high. In Formula 8B, theamount of palmitoyl tripeptide was increased to 3%, andphosphatidylserine (lipoid PS P 70) was added, along with oleuropein 80%at 0.025%. It was found that Lipoid PS P70 and oleuropein exhibited poorsolubility in heptyl undecylenate. In Formula 9B, the concentration ofheptyl undecylenate was lowered to 2.5%, caprylyl methicone was added tosolubilize the Lipoind PS P 70, which was increased to 0.05%, andoleuropein 80% was increased to 0.05%. The viscosity for Formula 9B wasapproximately 5000 cPs. The data demonstrated that caprylyl methiconewas effective in solubilizing the phosphatidylserine and oleuropein;however, the greater stability was obtained at the expense of viscosity.

Tests were conducted to evaluate the feasibility of adding additionalheptyl undecylenate to the formulations. In Formula 10B, theconcentration of heptyl undecylenate was increased from 2.5% to 8.75%.Formula 10B exhibited separation and instability, and had a viscosity ofapproximately 1800 cPs. In Formula 11B, the concentration of heptylundecylenate was increased from 8.75% to 16.75%. Formula 11B exhibitedseparation and instability, and had a viscosity of approximately 400cPs. The results of these tests confirmed that raising levels of heptylundecylenate resulted in instability of the formulations due tosolubility issues with phosphatidylserine and oleuropein despite thepresence of caprylyl methicone.

Other strategies were investigated for increasing viscosity whilemaintaining stability. In Formula 12B, the concentration of palmitoylhexapeptide-12 containing base was lowered from 5.5% to 3% with acorresponding increase in the cyclopentasiloxane, dimethiconecrosspolymer, yielding a formulation with a viscosity of approximately24000 cPs. In Formula 13B, the concentration of palmitoyl hexapeptide-12was lowered from 3% to 2% and heptyl undecylenate was removed toincrease viscosity and stability, resulting in a formulation having aviscosity of approximately 47000 cPs. In Formula 14B, the concentrationof palmitoyl hexapeptide-12 was increased back to 3% and the heptylundecylenate was removed, yielding a formulation having an approximateviscosity of 34000 cPs. The results of the testing demonstrated thatreducing the palmitoyl hexapeptide-12 containing base in favor of thecyclopentasiloxane, dimethicone crosspolymer increased viscosity to adesirable level even in the presence of heptyl undecylenate, resultingin a stable formulation.

A viscosity level of from 400 cPs to 50000 cPs is generally preferredfor topical formulations, for example, 1000 cPs to 30000 cPs, or 10000cPs to 25000 cPs, or 20000 cPs to 25000 cPs.

Formula 1B Ingredient % (wt.) Isododecane, Dimethicone Crosspolymer-395.468 Physalis Angulata Extract 1.000 Arnica Montana Extract 2.000Centella Asiatica Extract 0.250 Squalane 1.000 Growth Hormone ReleasingPeptide-2 0.032 1,3 Propanediol 0.250

Formula 2B Ingredient % (wt.) Isododecane, Dimethicone Crosspolymer-392.718 Panthenyl Triacetate/Naringenin 2.000 Arnica Montana Extract2.000 Dunaliella Salina Extract 1.000 Squalane 2.000 Growth HormoneReleasing Peptide-2 0.032 1,3 Propanediol 0.250

Formula 3B Ingredient % (wt.) Cyclopentasiloxane, Polysilicone-11 92.718Panthenyl Triacetate/Naringenin 2.000 Arnica Montana Extract 2.000Dunaliella Salina Extract 1.000 Squalane 2.000 Growth Hormone ReleasingPeptide-2 0.032 1,3 Propanediol 0.250

Formula 4B Ingredient % (wt.) Isododecane, Dimethicone Crosspolymer-387.500 Panthenyl Triacetate/Naringenin 2.000 Arnica Montana Extract2.000 Dunaliella Salina Extract 1.000 Squalane 2.000 PentaerythritylTetraisostearate, Caprylic/Capric 5.500 Triglyceride, StearalkoniumHectorite, Propylene Carbonate, Palmitoyl Hexapeptide-12

Formula 5B Ingredient % (wt.) Cyclopentasiloxane, Polysilicone-11 87.500Panthenyl Triacetate, Naringenin 2.000 Squalane, Dunaliella SalinaExtract 1.000 Glycine Soja (Soybean) Oil, Arnica Montana 2.000 Extract,Tocopherol Squalane 2.000 Pentaerythrityl Tetraisostearate,Caprylic/Capric 5.500 Triglyceride, Stearalkonium Hectorite, PropyleneCarbonate, Palmitoyl Hexapeptide-12

Formula 6B Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 84.500 Heptyl Undecylenate 5.000 Panthenyl Triacetate,Naringenin 2.000 Squalane, Dunaliella Salina Extract 1.000 Glycine Soja(Soybean) Oil, Arnica Montana 2.000 Extract, Tocopherol PentaerythritylTetraisostearate, Caprylic/Capric 5.500 Triglyceride, StearalkoniumHectorite, Propylene Carbonate, Palmitoyl Hexapeptide-12

Formula 7B Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 83.500 Heptyl Undecylenate 5.000 Panthenyl Triacetate,Naringenin 2.000 Squalane, Dunaliella Salina Extract 1.000 Glycine Soja(Soybean) Oil, Arnica Montana 2.000 Extract, Tocopherol PentaerythritylTetraisostearate, Caprylic/Capric 5.500 Triglyceride, StearalkoniumHectorite, Propylene Carbonate, Palmitoyl Hexapeptide-12 PentaerythritylTetraisostearate, Caprylic/Capric 1.000 Triglyceride, DisteardimoniumHectorite, Propylene Carbonate, Palmitoyl Tripeptide-1

Formula 8B Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 81.450 Squalane, Dunaliella Salina Extract 1.000 GlycineSoja (Soybean) Oil, Arnica Montana 2.000 Extract, TocopherolPentaerythrityl Tetraisostearate, Caprylic/Capric 5.500 Triglyceride,Stearalkonium Hectorite, Propylene Carbonate, Palmitoyl Hexapeptide-12Pentaerythrityl Tetraisostearate, Caprylic/Capric 3.000 Triglyceride,Stearalkonium Hectorite, Propylene Carbonate, Palmitoyl Tripeptide-1Heptyl Undecylenate 5.000 Phosphatidylserine, Phospholipids, Tocopherol,0.025 Ascorbyl Palmitate Panthenyl Triacetate, Naringenin 2.000 OleaEuropaea (Olive) Leaf Extract, Oleuropein 0.025

Formula 9B Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 83.40 Heptyl Undecylenate 2.500 Squalane, Dunaliella SalinaExtract 1.000 Glycine Soja (Soybean) Oil, Arnica Montana 2.000 Extract,Tocopherol Pentaerythrityl Tetraisostearate, Caprylic/Capric 5.500Triglyceride, Stearalkonium Hectorite, Propylene Carbonate, PalmitoylHexapeptide-12 Pentaerythrityl Tetraisostearate, Caprylic/Capric 3.000Triglyceride, Stearalkonium Hectorite, Propylene Carbonate, PalmitoylTripeptide-1 Caprylyl Methicone 0.500 Phosphatidylserine, Phospholipids,Tocopherol, 0.050 Ascorbyl Palmitate Panthenyl Triacetate, Naringenin2.000 Olea Europaea (Olive) Leaf Extract, Oleuropein 0.050

Formula 10B Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 77.15 Heptyl Undecylenate 8.750 Squalane, Dunaliella SalinaExtract 1.000 Glycine Soja (Soybean) Oil, Arnica Montana Extract, 2.000Tocopherol Pentaerythrityl Tetraisostearate, Caprylic/Capric 5.500Triglyceride, Stearalkonium Hectorite, Propylene Carbonate, PalmitoylHexapeptide-12 Pentaerythrityl Tetraisostearate, Caprylic/Capric 3.000Triglyceride, Stearalkonium Hectorite, Propylene Carbonate, PalmitoylTripeptide-1 Caprylyl Methicone 0.500 Phosphatidylserine, Phospholipids,Tocopherol, Ascorbyl 0.050 Palmitate Panthenyl Triacetate, Naringenin2.000 Olea Europaea (Olive) Leaf Extract, Oleuropein 0.050

Formula 11B Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 69.150 Heptyl Undecylenate 16.750 Squalane, DunaliellaSalina Extract 1.000 Glycine Soja (Soybean) Oil, Arnica Montana 2.000Extract, Tocopherol Pentaerythrityl Tetraisostearate, Caprylic/Capric5.500 Triglyceride, Stearalkonium Hectorite, Propylene Carbonate,Palmitoyl Hexapeptide-12 Pentaerythrityl Tetraisostearate,Caprylic/Capric 3.000 Triglyceride, Stearalkonium Hectorite, PropyleneCarbonate, Palmitoyl Tripeptide-1 Caprylyl Methicone 0.500Phosphatidylserine, Phospholipids, Tocopherol, 0.050 Ascorbyl PalmitatePanthenyl Triacetate, Naringenin 2.000 Olea Europaea (Olive) LeafExtract, Oleuropein 0.050

Formula 12B Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 85.900 Heptyl Undecylenate 2.500 Squalane, DunaliellaSalina Extract 1.000 Glycine Soja (Soybean) Oil, Arnica Montana 2.000Extract, Tocopherol Pentaerythrityl Tetraisostearate, Caprylic/Capric3.000 Triglyceride, Stearalkonium Hectorite, Propylene Carbonate,Palmitoyl Hexapeptide-12 Pentaerythrityl Tetraisostearate,Caprylic/Capric 3.000 Triglyceride, Stearalkonium Hectorite, PropyleneCarbonate, Palmitoyl Tripeptide-1 Caprylyl Methicone 0.500Phosphatidylserine, Phospholipids, Tocopherol, 0.050 Ascorbyl PalmitatePanthenyl Triacetate, Naringenin 2.000 Olea Europaea (Olive) LeafExtract, Oleuropein 0.050

Formula 13B Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 89.40 Squalane, Dunaliella Salina Extract 1.000 GlycineSoja (Soybean) Oil, Arnica Montana 2.000 Extract, TocopherolPentaerythrityl Tetraisostearate, Caprylic/Capric 2.000 Triglyceride,Stearalkonium Hectorite, Propylene Carbonate, Palmitoyl Hexapeptide-12Pentaerythrityl Tetraisostearate, Caprylic/Capric 3.000 Triglyceride,Stearalkonium Hectorite, Propylene Carbonate, Palmitoyl Tripeptide-1Caprylyl Methicone 0.500 Phosphatidylserine, Phospholipids, Tocopherol,0.050 Ascorbyl Palmitate Panthenyl Triacetate, Naringenin 2.000 OleaEuropaea (Olive) Leaf Extract, Oleuropein 0.050

Formula 14B Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 88.70 Squalane, Dunaliella Salina Extract 1.000 GlycineSoja (Soybean) Oil, Arnica Montana 2.000 Extract, TocopherolPentaerythrityl Tetraisostearate, Caprylic/Capric 3.000 Triglyceride,Stearalkonium Hectorite, Propylene Carbonate, Palmitoyl Hexapeptide-12Pentaerythrityl Tetraisostearate, Caprylic/Capric 3.000 Triglyceride,Stearalkonium Hectorite, Propylene Carbonate, Palmitoyl Tripeptide-1Caprylyl Methicone 0.250 Phosphatidylserine, Phospholipids, Tocopherol,0.025 Ascorbyl Palmitate Panthenyl Triacetate, Naringenin 2.000 OleaEuropaea (Olive) Leaf Extract, Oleuropein 0.025

Example 1C

Exemplary formulations are prepared.

Exemplary Formula 1C Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer  82-92 Palmitoyl hexapeptide-12 0.01-0.1 Palmitoyltripeptide-1 0.01-0.1 Other Components remainder

Exemplary Formula 2C Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 82-92 Heptyl Undecylenate 1-4 Palmitoyl hexapeptide-120.01-0.1  Palmitoyl tripeptide-1 0.01-0.1  Other Components remainder

Exemplary Formula 3C Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer  82-92 Palmitoyl hexapeptide-12 0.01-0.1 Palmitoyltripeptide-1 0.01-0.1 Caprylyl methicone 0.25-1   Phospatidyl serine0.05-0.1 Oleuropein 0.05-0.1 Other Components remainder

Exemplary Formula 4C Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer  82-92 Caprylyl methicone 0.25-1   Phospatidyl serine0.05-0.1 Oleuropein 0.05-0.1 Other Components remainder

Exemplary Formula 5C Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer  82-92 Heptyl Undecylenate  1-4 Palmitoyl hexapeptide-120.01-0.1 Palmitoyl tripeptide-1 0.01-0.1 Caprylyl methicone 0.25-1  Phospatidyl serine 0.05-0.1 Oleuropein 0.05-0.1 Other Componentsremainder

Exemplary Formula 6C Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer  82-92 Heptyl Undecylenate  1-4 Carrier containingPalmitoyl hexapeptide-12 (@100 ppm) 0.5-6  Carrier containing Palmitoyltripeptide-1 (@100 ppm) 0.5-6  Caprylyl methicone 0.25-1   Phospatidylserine 0.05-0.1 Oleuropein 0.05-0.1 Other Components remainder

Exemplary Formula 7C Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 82-92 Heptyl Undecylenate 1-4 Carrier: 2-6  Pentaerythrityltetraisostearate  Caprylic/capric triglyceride  Propylene carbonate Stearalkonium hectorite Palmitoyl hexapeptide-12** Carrier: 1-6 Pentaerythrityl tetraisostearate  Caprylic/capric triglyceride Propylene carbonate  Disteardimonium hectorite Palmitoyl tripeptide-1**Caprylyl methicone 0.25-1   Phospatidyl serine/lecithin 0.05-0.1 Oleuropein 0.05-0.1  Other Components remainder *Present in carrier at100 ppm. **Present in carrier at 100 ppm.

Exemplary Formula 8C Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 82-92 Heptyl Undecylenate 1-4 Carrier: 1-6  Pentaerythrityltetraisostearate  Caprylic/capric triglyceride  Propylene carbonate Stearalkonium hectorite Palmitoyl hexapeptide-12* Carrier: 1-6 Pentaerythrityl tetraisostearate  Caprylic/capric triglyceride Propylene carbonate  Disteardimonium hectorite Palmitoyl tripeptide-1**Caprylyl methicone 0.25-1   Phospatidyl serine/lecithin 0.05-0.1 Oleuropein 0.05-0.1  Other Components 3.5-14  PanthenylTriacetate/Naringenin (or other anti-irritant) 1-4 Arnica MontanaExtract (or other anti-inflammatory) 1-4 Dunaliella Salina Extract (orother antioxidant) 0.5-2   *Present in carrier at 100 ppm. **Present incarrier at 100 ppm.

Exemplary Formula 9C Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 82-92 Heptyl Undecylenate 1-4 Carrier containing Palmitoylhexapeptide-12 (100 ppm) 2-5 Carrier containing Palmitoyl tripeptide-1(100 ppm) 2-5 Caprylyl methicone 0.25-1   Phospatidyl serine/lecithin0.05-0.1  Oleuropein 0.05-0.1  Other Components 3.5-14 

Exemplary Formula 10C Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 82-92 Heptyl Undecylenate 1-4 Carrier: 1-6  Pentaerythrityltetraisostearate  Caprylic/capric triglyceride  Propylene carbonate Stearalkonium hectorite Palmitoyl hexapeptide-12* Carrier: 1-6 Pentaerythrityl tetraisostearate  Caprylic/capric triglyceride Propylene carbonate  Disteardimonium hectorite Palmitoyl tripeptide-1**Caprylyl methicone 0.25-1   Phospatidyl serine/lecithin 0.05-0.1 Oleuropein 0.05-0.1  Other Components 3.5-14 

Exemplary Formula 11C Ingredient % (wt.) Cyclopentasiloxane, DimethiconeCrosspolymer 82-92 Heptyl Undecylenate 1-4 Pentaerythrityltetraisostearate 2-5 Caprylic/capric triglyceride Propylene carbonateStearalkonium hectorite Palmitoyl hexapeptide-12 Pentaerythrityltetraisostearate 2-5 Caprylic/capric triglyceride Propylene carbonateDisteardimonium hectorite Palmitoyl tripeptide-1 Caprylyl methicone0.25-1   Phospatidyl serine/lecithin 0.05-0.1  Oleuropein 0.05-0.1 Other Components 3.5-14  Anti-irritant 1-4 Anti-inflammatory 1-4Anti-oxidant 0.5-2  

Example 2: Laser Wound Study

A laser wound study was conducted wherein subjects received a 3 mmerbium CO₂ laser spot on his or her forearm. Wound healing progress wasobserved for 18 days post-wound. Half of the subjects treated theirwound at least one time a day for 18 days using a topical formulation(referred to herein as “a dual peptide”) containing 1.0 wt. % GKH and5.5 wt. % VGVAPG in an anhydrous silicone elastomer gel matrix. Theother half of the subjects followed the same post-wound treatmentprotocol, but using a control formulation instead of a dual peptideformulation. The control was either Aquaphore® (comprising petrolatum,panthenol, glycerin, and bisabolol) from Beiersdorf Inc., Wilton, Conn.,or no treatment). In FIG. 4A, a graph depicts the wound appearance forsubjects receiving a dual peptide treatment versus control treatmentover the course of 18 days post-wound. Wound appearance was superior forsubjects receiving a dual peptide treatment, with significantdifferences on Days 2 to 14 when compared to the control. In FIG. 4B,the data demonstrates epithelial confluence was superior for subjectsreceiving a dual peptide treatment from Days 2 to 18, with noticeabledifferences between the two treatments on each of those days. The datadepicted in FIG. 4C shows there was less crusting/scabbing in subjectsreceiving a dual peptide treatment from Days 1 to 18, with significantdifferences from Days 1 to 14.

Example 3: Laser Wound Study

A study following the same protocol as Example 2 was conducted, whichincluded testing of a commercial wound care treatment (Aquaphor®) inaddition to a dual peptide and control. FIG. 5A providescrusting/scabbing data demonstrating that a dual peptide treatment wassuperior to the control from Days 1 to 18 and superior to Aquaphor® onDays 2 through 11. FIGS. 5B-D are photographs of wound appearance on Day4 for three different wounds, each treated with either a dual peptide(FIG. 5B), the control treatment (FIG. 5C), or Aquaphor® (FIG. 5D). Thephotographs demonstrate that a dual peptide treatment exhibits the leastamount of crusting or scabbing over the wound.

Example 4: Laser Wound Study

A laser wound study was conducted wherein subjects received a 1 mmerbium CO₂ laser spot on his or her forearm, the spot penetratingthrough the epidermis and deep into the dermis. The subjects receivedlocal anesthesia injections prior to receiving the laser wound. Eachsubject applied a dual peptide multiple times per day for two weeks(Pre-Wound Days 1-14) prior to receiving the laser wound. Half of thesubjects received a dual peptide treatment at least one time per day for9 days following receiving the laser wound, and the other half of thesubject received Aquaphor® treatment multiple times per day for 9 daysfollowing receiving the laser wound. FIG. 6A-B provides data pertainingto wound appearance (FIG. 6A) and crusting/scabbing (FIG. 6B),demonstrating the wound healing benefits of two weeks of pretreatmentwith a dual peptide prior to a laser skin procedure and treatment witheither a dual peptide or Aquaphor® post-procedure. In FIG. 6A, woundappearance data is provided for a dual peptide versus Aquaphor®treatment from Post-Wound Days 1 through Days 4, 7, and 9, with ameaningful difference on Post-Wound Day 9. In FIG. 6B, the differencesin crusting and scabbing for a dual peptide treatment versus Aquaphortreatment is compared for Post-Wound Day 1 through Day 4, 7, and 9, withnoticeable differences on Days 1 through 4. FIGS. 6C-D providephotographed wound images for the Post-Wound a dual peptide treatment(FIG. 6C) and Post-Wound Aquaphor® treatment (FIG. 6D) on Day 9,demonstrating superior wound healing associated with a dual peptidepre-treatment and post-treatment.

Example 5: Laser Esthetic Treatment Study

A human subject received a facial aesthetic laser treatment. The lasertreatment utilized an Encore UltraPulse® ActiveFX™ CO₂ laser. Thetreatment protocol included a dual peptide pre-treatment for 21 daysprior to a facial aesthetic laser treatment. Post-procedure, the patientwas treated with a dual peptide on Days 1-14 (“invasive kit”, asdescribed herein) and from Day 14 onward (“noninvasive kit”, asdescribed herein). The photographs demonstrate images of the patientprior to treatment (FIG. 7A), Day 4 post-laser treatment (FIG. 7B), andDay 9 post-laser treatment (FIG. 7C). By Day 9 post-laser treatment(post-procedure), the patient's skin appears to have fully recoveredfrom the procedure.

Example 6: Collagen and Elastin mRNA Study

Fibroblast monolayers were exposed to a dual peptide for 48 hours. mRNAproduction was compared to that of a set of fibroblast cells that weresubjected to exposure to a control. After 48 hours, the fibroblastmonolayers were harvested and mRNA was extracted from the cell lysate.Fibroblasts that were subjected to a dual peptide had elastin (ELN) mRNAlevels more than three-fold greater than the control fibroblasts. Thedual peptide also stimulated the fibroblasts to produce a more thantwo-fold increase in collagen (COL1A1) mRNA production compared to thecontrol. FIG. 8 depicts the results of a study where fibroblastmonolayers were exposed to a dual peptide composition. One set offibroblasts were exposed to a dual peptide for 48 hours and a second setreceived a control treatment for 48 hours. The cells were then harvestedand mRNA extracted from the cell lysates. The level of elastin (ELN)mRNA was more than 3 times higher for the dual peptide treated cellsthan the control fibroblasts. Collagen (COL1A1) mRNA production was overtwice as high as the control fibroblasts. The data demonstrate that adual peptide significantly upregulates collagen and elastin mRNA.

Example 7: Preconditioning

The concept of pre-conditioning is akin to the wound bed preparationconcept practiced in wound healing. Preconditioning can be described asskin bed preparation. In order to maximize the healing capacity oftherapeutic agents in chronic wounds, the bed must be debrided andprepared, ensuring that concentrations of proteases and cytokines areleveled to avoid lysis and degradation of these therapeutic agents fromwithin the wound bed or from the corrosive wound fluid produced by thesechronic wounds. See, e.g., Widgerow A D. Chronic wound fluid—thinkingoutside the box. Wound repair and regeneration: official publication ofthe Wound Healing Society [and] the European Tissue Repair Society 2011;19(3): 287-91.

In much the same way, chronic photoaged skin needs adequate preparationto ensure the therapeutic procedure is optimized in its regenerativecapacity. However, the background milieu is different. Beforeregeneration can take place, modulation of the extracellular matrix(ECM) must occur. In the case of damaged skin, this normally involvescombatting the excessive fragmentation and agglutination of ECMproteins, primarily collagen, elastin and some glycosaminoglycans, whichhave collected in disorganized bundles resistant to protease digestion.The result is dysfunctional cell to cell and cell to matrix cross-talkand inefficient ongoing repair. See, e.g., Fligiel S, Varani J, Datta S,Kang S, Fisher G, Voorhees J J. Collagen Degradation inAged/Photodamaged Skin InVivo and After Exposure to MatrixMetalloproteinase-1 InVitro. The Journal of investigative dermatology2003; 120: 842-8. The resurfacing procedure usually results indenaturation of collagen producing extra fragmentation and an increasedload for the ECM.

The theory of pre-conditioning is based upon the premise that aiding theprocess of degradation of these ECM bundles aids in clearing the ECMthus improving cellular and matrix cross talk. This allows theregenerative phase of repair to occur earlier in the process thusencouraging more efficient healing.

Denatured collagen and elastin fragments present as gelatins and themajor proteases involved in clearing these fragments aremetalloproteinases (MMP) 2 and 9, the gelatinases. See, e.g., Simeon A,Monier F, Emonard H, et al. Expression and Activation of MatrixMetalloproteinases in Wounds: Modulation by the Tripeptide-CopperComplex Glycyl-L-Histidyl-L-Lysine-Cu2F. The Journal of investigativedermatology 1999; 112: 957-62. A balance of MMP function and activity isextremely important for normal wound healing—chronic wounds exhibit anover-abundance of MMP activity with destruction of much of the ECMincluding the matrikine fragments derived from collagen and elastin thatstimulate regeneration. Thus, pre-conditioning is aimed at temporarilyincreasing levels of MMPs (2 and 9 in particular) in order to degradeECM fragment bundles and then allowing the newly denatured collagenfragments to stimulate regeneration in a relatively cleared ECM.

It is apparent that a balance of inflammation and protease activity issought and the tri-hex technology introduces increased levels of MMPs 2and 9 which are secreted at different times in the healing process.Studies have shown that GHK is able to modulate MMP expression andactivation in wounds. MMP-2 was detected in wound models increasing fromday 3 to day 22 after wound chamber implantation, whereas MMP 9expression increase is short-lived coinciding with neutrophilinfiltration, monocyte and macrophage activity and early phagocytosis.MMP2 then continues to clear the matrix over the next 3 weeks or so,clearing a pathway for angiogenesis and revascularization. FIG. 9includes a graph showing gelatinolytic activity (peaks area/μg protein)over time for MMP-2. The dotted line is for GHK application to the woundchamber versus a control (solid line). GHK activates MMP-2 (gelatinase,Y axis) to far greater degree than control from day 3 and peaking atdays 18-22. FIG. 10 includes a graph showing gelatinolytic activity(peaks area/μg protein) over time. The dotted line is for GHKapplication to the wound chamber versus a control (solid line). GHKactivates MMP-2 (gelatinase, Y axis) to far greater degree than controlfrom day 12 and peaking at days 18-22. See Simeon et al., J InvestDermatol 112: 957-964, 1999.

Peptides, such as the tripeptides disclosed herein, stimulate increasedlevels of MMP-2, associated with extracellular matrix clearance beforeregeneration of collagen and elastin. There is also a direct correlationbetween MMP-2 and adipogenesis—the generation of new healthy fat.Adipogenesis is advantageous in that it can provide a volumizing effectfor anti-aging, skin plumping and freshening effects. Adipose tissue(AT) expansion and regression are well-controlled processes in responseto nutritional status. Expansion consists of hypertrophy, where existingadipocytes increase in volume, and hyperplasia, where newly formedadipocytes differentiate from precursor cells (adipogenesis). Tissuegrowth is also associated with angiogenesis and intensive proteolyticremodeling of the extracellular matrix (ECM). All of these processesare, in part, regulated by the matrix metalloproteinase (MMP) system.MMP deficiency has been observed to be associated with impaired ATdevelopment. Several studies show that the gelatinase subgroup (MMP-2and MMP-9) is secreted by AT and that their activity is modulated duringAT expansion/regression. Elevated plasma levels of MMP-2 are observed inobese patients, and these levels drop after bariatric surgery. Based onin vitro studies performed with rat, mouse (3T3-F442A and 3T3-L1 celllines) and human preadipocytes, a role for the gelatinases has beensuggested in differentiation of adipocytes.

The role of MMP-2 in in vitro preadipocyte differentiation, and in vivode novo fat pad formation using precursor cells with genetic deficiency,gene silencing or overexpression of MMP-2 has been demonstrated. SeeBauters et al., Biochimica et Biophysica Acta 1850 (2015) 1449-1456.Bauters et al. utilized an in vivo model of de novo adipogenesis wherein3T3-F442A preadipocytes transduced with MMP-2 shRNA constructTRCN0000031228 or SHC002V negative control were injected subcutaneouslyin the back of 8 week-old male athymic BALB/cNude mice. Bauters et al.examined the size and density of adipocytes, as well as blood vesseldensity normalized to adipocyte number and observed the differentiationof embryonic fibroblasts into mature adipocytes. A significant increasein lipid accumulation during differentiation of 3T3-F442A cells withMMP-2 overexpression was observed, and was supported by significantlyincreased expression of aP2 and Ppar-γ. The de novo fat pads formedexhibited no differences in adipocyte size and density.

Differentiation of preadipocytes into new mature adipocytes can bedivided in two main phases. The determination phase consists ofcommitment of progenitor cells to the adipocyte lineage. In the terminaldifferentiation phase, preadipocytes acquire the specificcharacteristics of mature adipocytes. By reducing the levels of MMP-2via selective gene silencing, the differentiation of 3T3-F442A cells isreduced compared to control cells. MMP-2 overexpression had the opposedeffect and stimulated differentiation of 3T3-F442A preadipocytes.However, even with minimally enhanced MMP-2 levels, enhanceddifferentiation was observed. Thus, MMP-2 appears to play a functionalrole in early and later stages of adipogenesis in vitro. Endothelialcells produce and secrete MMPs and it has been reported that in vivoadipogenesis is dependent on angiogenesis. Thus, local levels of activeMMP-2 (independent of its origin) may be sufficient to account for itsrole in adipogenesis. Accordingly, by administering peptide, such as thetripeptides of the embodiments, to a patient, increased levels of MMP-2can be obtained, which in turn can stimulate adipogenesis. Thestimulated adipogenesis can yield a volumizing effect, resulting in thereduction in appearance of signs of aging, as well as plumping andfreshening of the skin.

The second area relating to ECM preparation involves the process ofangiogenesis, the basis of nutrient delivery and exchange. The processis linked to the stimulation of VEGF and vessel development and growthtakes weeks to develop. Pre-vascularization is expected to optimizewound healing. It has been demonstrated that the delivery modality forinducing angiogenesis must provide an extended (4 to 6 weeks) presenceof the therapeutic agent at the site of desired vessel growth. See,e.g., Simons M. Angiogenesis: where do we stand now? Circulation 2005;111(12): 1556-66. Such a prolonged presence cannot be achieved withsingle-dose administration of proteins or peptides but needs aprotracted period of application. GHK dose-dependently increases thesecretion of VEGF and stimulate tubular formation and neovascularizationin animal models. FIG. 11 shows VEGF concentration (pg VEGF/ng DNA) as afunction of GHK (ng/mL) concentration. See, e.g., Jose S, Hughbanks M L,Binder B Y, Ingavle G C, Leach J K. Enhanced trophic factor secretion bymesenchymal stem/stromal cells with Glycine-Histidine-Lysine(GHK)-modified alginate hydrogels. Acta biomaterialia 2014; 10(5):1955-64. Thus, a 4 week start to the process of administering a dualpeptide prior to laser treatment ensures a robust start to angiogenicstimulation of the ECM.

To assess the effect of pre-conditioning with dual peptides, a subjectapplied dual peptides to the fore arm for 2-3 weeks, and then a biopsywas taken from the treated fore arm as well as the another from theuntreated (control) fore arm. The results are seen in FIGS. 12A and 12B.FIG. 12A shows the baseline, untreated sample biopsy stained with H/E.The baseline sample (FIG. 12A) shows irregularly clumped collagen in adisorganized extracellular matrix; the basal cells in thedermo-epidermal junction are flat displaying reduced functionality.After 2-3 weeks treatment with dual peptide, the biopsy (FIG. 12B) showsan extracellular matrix that has been cleared of a considerable amountof clumped collagen bundles and replaced with a more organizeddistribution and arrangement of the collagen; the basal stem cells atthe dermo-epidermal junction are cuboidal in shape representing morefunctional activity and the epidermis is thicker with a healthierappearance.

Pre-conditioning is therefore a useful process for optimizing woundhealing and esthetic outcomes on patients undergoing resurfacingprocedures. This is based on the concept of wound bed preparationensuring an early start to ECM remodeling. More particularly, earlyMMP-9 and later ongoing MMP-2 upregulation facilitates degradation ofaccumulated ECM protein bundles seen in elastotic photo-aged skin. Inaddition, stimulating angiogenesis on the basis of VEGF stimulation overa period of time improves ECM status in relation to future woundhealing. A 2-3 week period is generally a minimum time satisfactory forpre-conditioning, but in certain circumstances a shorter preconditioningtime can be employed. Accordingly, pre-conditioning for at least the twoweek period leading up to a procedure can advantageously be employed,e.g., pre-conditioning for two weeks, three weeks, four weeks or more,e.g., 2-4 weeks. Daily preconditioning is desirable, e.g.,administration at least once a day, e.g., 2, 3, 4, or more times a day.Twice daily administration, e.g., morning and evening, can convenientlybe employed. In certain embodiments, less than once per day applicationcan be acceptable, e.g., every other day or every three days, or twiceweekly.

Similar to pre-conditioning, post-treatment use can also beadvantageously employed. A 2 week period is generally a minimum timesatisfactory for post-treatment use, but in certain circumstances ashorter post-treatment use time can be employed. Accordingly,post-treatment use for at least the two week period following aprocedure, once the skin is sufficiently healed such that the topicalformulation can be applied, can be advantageously employed, e.g.,post-treatment for two weeks, three weeks, four weeks or more, e.g., 2-4weeks. Daily post-treatment administration is desirable, e.g.,administration at least once a day, e.g., 2, 3, 4, or more times a day.Twice daily administration, e.g., morning and evening, can convenientlybe employed. In certain embodiments, less than once per day applicationcan be acceptable, e.g., every other day or every three days, or twiceweekly.

In some instances, maintenance treatment of indefinite duration can bedesirable to promote skin health, skin repair, and/or optimal skin bedcondition. For maintenance, it may be acceptable to administer lessfrequently than once a day, e.g., once a week, or once every two orthree days; however, twice daily administration, e.g., morning andevening, can conveniently be employed.

When the peptides are formulated in combination with other therapeuticagents, the formulation can be applied according to the preferredadministration protocol for the other therapeutic agent(s).Alternatively, the administration protocol may be that as presentedherein for compositions wherein the peptides are the only therapeuticagents present.

In various embodiments, various combinations of one or more ofpre-conditioning, post-treatment application, and maintenance treatmentcan be employed.

Example 8: Elastin Production

The impact of the dual peptide formulation on elastin production wasinvestigated. A subject applied the dual peptide to a left forearm dailyfor 2-3 weeks and biopsies were obtained at of the conclusion of thedaily treatment. Skin biopsy samples were taken from the treated portionof the left fore arm (dual peptide, FIG. 13B), and from an untreatedportion of the right forearm (control, FIG. 13A). Usingimmune-histochemistry (IHC) techniques, the presence of elastin proteinis seen as brown staining in the photographs at 20× and 100×magnification that were obtained. As the photographs show, a significantincrease in elastin protein levels was observed for skin treated withthe dual peptide formulation versus the control. A significantly lowerlevel of elastin was observed in the untreated portion.

Example 9: Increased Pro-Collagen Levels

The impact on pro-collagen in dual peptide treated skin wasinvestigated. Prior to the initiation of treatment with dual peptides, abaseline biopsy of the facial skin was obtained. The subject thenapplied the dual peptide to their face daily for 2 weeks and a secondbiopsy was obtained. FIG. 14A is a skin biopsy photograph (100×) showinglow staining for pro-collagen by IHC as a baseline condition while FIG.14B is a skin biopsy photograph (100×) showing significantly increasedlevels of pro-collagen after 2 weeks of dual peptide treatment.

Example 10: Increased Collagen

FIGS. 15A-B provide photographs (100×) of skin biopsy samples for dualpeptide treated skin three weeks after application (FIG. 15B) and abaseline sample (FIG. 15A). Upper dermal increased collagen formationover three weeks of topical application was observed, with solarelastosis pushed down by new collagen and improved epidermis appearance.

Example 11: Distribution of Elastin

FIGS. 16A-C provide photographs (100×) of skin biopsy samples for dualpeptide treated facial skin three weeks after application (FIG. 16B),eight weeks after application (FIG. 16C) and a baseline sample (FIG.16A). In clients who have elastin in elastotic photodamaged elastictissue, topical application of the dual peptide results in the elastinmaterial to be less clumped and markedly distributed into deeper dermallayers over the eight week period.

Example 12: Decreased MMP1

FIGS. 17A-D provide photographs of skin biopsy samples for dual peptidetreated skin three weeks after application at 40× (FIG. 17B), a baselinesample at 40× (FIG. 8A), a skin biopsy sample for dual peptide treatedskin three weeks after application at 100× (FIG. 17D) and a baselinesample at 100× (FIG. 17C). Decreased MMP1 staining was observed over thethree week period in the pre-auricular region.

Example 13: Increased Decorin

FIGS. 18A-B provide photographs (100×) of skin biopsy samples for dualpeptide treated skin three weeks after application (FIG. 18B) and abaseline sample (FIG. 18A). Increased decorin staining was observed overthe three week period in the pre-auricular region. Decorin is believedto influence fibrillogenesis, and also interacts with fibronectin,thrombospondin, the complement component C1q, epidermal growth factorreceptor (EGFR) and transforming growth factor-beta (TGF-beta). Decorinhas been shown to either enhance or inhibit the activity of TGF-beta 1.The primary function of decorin involves regulation during the cellcycle.

Example 14: Comparison of Dual Peptide Formulation to Standard of Care

To evaluate the efficacy in accelerating wound healing and subjectsatisfaction of Formulation 16A (composition provided above), it wascompared to standard of care (SOC) following intense pulsed light (IPL)and/or pulsed dye laser (PDL) with Q-switch-alexandrite and fractionatedCO₂ laser resurfacing compared to Vaniply regimen (PharmaceuticalSpecialties, Inc., Rochester, Minn.). In addition, an analysis ofadverse events was undertaken as the treatment formulations differed—abland preservative free preparation (Vaniply) versus the otherpreparation containing active peptides and botanicals (Formulation 16A).

In this single-blind, randomized study, 15 female subjects aged 45-70years with Fitzpatrick skin types I-III and moderate to severephotodamage underwent IPL and/or PDL with Q-switch-alexandrite andfractionated 10,600 nm CO₂ laser resurfacing of the face. Subjects wererandomized on a 2:1 ratio to Formulation 16A or the standard of care(Vaniply ointment regimen). The subjects were pre-treated 3 weeks priorto resurfacing and continued for 8 weeks after the procedure. Subjectswere evaluated on postoperative days 1, 3, 4, 7, 28, and 84. Endpointsincluded investigator- and subject-rated signs and symptoms of healingand subject-rated satisfaction questionnaire (p<0.05).

Assessment of healing in the first 2 week period post procedure wasconsidered the most important end point. The overall analysis frominvestigator reporting was that skin healing and patient experience overthe first 7 days (and all time points up to that time) post-resurfacinglaser treatment was superior in the Formulation 16A group compared toexpected healing outcomes. The Formulation 16A group showed better skinhealing than the standard of care in all time intervals and by 7 daysthe group showed statistically significant advanced healing comparedwith SOC (P=0.015). In addition the blinded investigator-rated healingrating and reported patient experience was higher in the Formulation 16Agroup than in the SOC group for all of the post-procedure timepoints,once again with statistical significance by day 7 (P=0.02). Theturnaround time for healing and the day when differences between thegroups began to manifest most obviously was day 3, when signs such aserythema (P=0.02) and exudation (P=0.01), and symptoms such as skintenderness (P=0.02), burning and stinging (P=0.03) showed statisticallysignificant differences between the 2 groups. As time progressed in thestudy, subjective assessment improved and by study end (day 84)significantly better results were obtained in the Formulation 16A grouppertaining to the following 3 statements:

-   -   “Made me feel more confident in the way my skin looks” on Day 28        (P=0.08) and Day 84 (P=0.02)    -   “I would continue using this treatment regimen” on Day 28        (P=0.08) and Day 84 (P=0.03)    -   “I would recommend this treatment to others” on Day 28 (P=0.08)        and Day 84 (P=0.03).

Formulation 16A appeared to produce improvements in the healingexperience following IPL and/or PDL with Q-switch-alexandrite andfractionated CO₂ laser resurfacing of the face. The apparent reductionin healing time with Formula 16A allowed subjects to resume their normalactivities of daily living sooner. In addition, subjects were moresatisfied with Formula 16A than standard of care and without any reportof adverse events.

Example 21: Comparison of Dual Peptide Formulation to Standard of Care

A study was conducted to evaluate the efficacy in accelerating woundhealing and subject satisfaction of the Alastin Procedure EnhancementSystem (“Alastin”, or Formula 16A) compared to standard of carefollowing IPL and/or PDL with Q-switch-alexandrite and fractionated CO₂laser resurfacing (Pharmaceutical Specialties, Inc., Rochester, Minn.)compared to Vaniply regimen (standard of care) particularly in the firstweek of treatment. The aim was to assess objectively if the Alastingroup healed faster within the 7-day period, suffered any adverseevents, experienced greater symptomatic relief over the same period andwhether clients were able to enter the work force or society earlierthan SOC group. In summary, the study was an assessment of downtime andpatient experience following aggressive resurfacing procedures.Endpoints included investigator- and subject-rated signs and symptoms ofhealing and subject-rated comfort with procedure and satisfactionquestionnaire.

In analyzing the results of the trial, in view of relatively smallnumbers the one sided Student-t test was used with statisticalsignificance defined as ≤0.05.

Statistically Significant Differences

The overall analysis from investigator reporting was that skin healingand patient experience over the first 7 days (and all time points up tothat time) post-resurfacing laser treatment was superior in the Alastingroup compared to expected healing outcomes. The Alastin group showedbetter skin healing than the standard of care in all time intervals andby 7 days the group showed statistically significant advanced healingcompared with SOC (P=0.015). In addition the blinded Investigator-ratedhealing rating and reported patient experience was higher in the Alastingroup than in the SOC group for all of the post-procedure timepoints,once again with statistical significance by day 7 (P=0.02). The resultsof the Investigator Questionnaire including Healing Rating andExperience are presented in FIG. 19A and FIG. 19B, respectively.

The turnaround time for healing and the day when differences between thegroups began to manifest most obviously was day 3, when signs such aserythema (P=0.02) and exudation (P=0.01), and symptoms such as skintenderness (P=0.02), burning and stinging (P=0.03) showed statisticallysignificant differences between the two groups. The results of analysisof signs post procedure on day 3, symptoms post procedure on day 3, andsubject satisfaction are presented in FIG. 19C, FIG. 19D, and FIG. 19E,respectively.

On subjective scoring, the Alastin group consistently demonstratedsuperior outcomes. The Alastin group showed statistically significanthigher long term subject satisfaction than the SOC reaching significantlevels by Day 84 (P=0.03). For the Subject Global Aesthetic Improvementof long term Skin Quality, by the end of the study (day 84), the Alastingroup had statistically significant better results than the SOC groupfor the following 3 statements:

-   -   “Made me feel more confident in the way my skin looks”—Day 84        (P=0.02) (Question 11)    -   “I would continue using this treatment regimen”—Day 84 (P=0.03)        (Question 12)    -   “I would recommend this treatment to others”—Day 84 (P=0.03)        (Question 13)

The results of the answers to Questions 11, 12, and 13 are presented inFIG. 19F, FIG. 19G, and FIG. 19H, respectively.

No Significant Differences

Investigator Evaluation of Side Effects—Crusting: Crusting was Worse forthe SOC group on Days 1, 4 and 7; however worse for the Alastin group onDay 3. Due to the healing properties of the Alastin OcclusiveMoisturizer, the Crust has a better adherence to the face and comes offbetween Days 3 and 4, thus day 4 shows a dramatic reversal with a 35%improved appearance over SOC at day 4. The Investigator evaluation ofside effects—crusting are presented in FIG. 19I.

The above description presents the best mode contemplated for carryingout the present invention, and of the manner and process of making andusing it, in such full, clear, concise, and exact terms as to enable anyperson skilled in the art to which it pertains to make and use thisinvention. This invention is, however, susceptible to modifications andalternate constructions from that discussed above that are fullyequivalent. Consequently, this invention is not limited to theparticular embodiments disclosed. On the contrary, this invention coversall modifications and alternate constructions coming within the spiritand scope of the invention as generally expressed by the followingclaims, which particularly point out and distinctly claim the subjectmatter of the invention. While the disclosure has been illustrated anddescribed in detail in the drawings and foregoing description, suchillustration and description are to be considered illustrative orexemplary and not restrictive.

All references cited herein are incorporated herein by reference intheir entirety. To the extent publications and patents or patentapplications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

Unless otherwise defined, all terms (including technical and scientificterms) are to be given their ordinary and customary meaning to a personof ordinary skill in the art, and are not to be limited to a special orcustomized meaning unless expressly so defined herein. It should benoted that the use of particular terminology when describing certainfeatures or aspects of the disclosure should not be taken to imply thatthe terminology is being re-defined herein to be restricted to includeany specific characteristics of the features or aspects of thedisclosure with which that terminology is associated. Terms and phrasesused in this application, and variations thereof, especially in theappended claims, unless otherwise expressly stated, should be construedas open ended as opposed to limiting. As examples of the foregoing, theterm ‘including’ should be read to mean ‘including, without limitation,’‘including but not limited to,’ or the like; the term ‘comprising’ asused herein is synonymous with ‘including,’ ‘containing,’ or‘characterized by,’ and is inclusive or open-ended and does not excludeadditional, unrecited elements or method steps; the term ‘having’ shouldbe interpreted as ‘having at least;’ the term ‘includes’ should beinterpreted as ‘includes but is not limited to;’ the term ‘example’ isused to provide exemplary instances of the item in discussion, not anexhaustive or limiting list thereof; adjectives such as ‘known’,‘normal’, ‘standard’, and terms of similar meaning should not beconstrued as limiting the item described to a given time period or to anitem available as of a given time, but instead should be read toencompass known, normal, or standard technologies that may be availableor known now or at any time in the future; and use of terms like‘preferably,’ ‘preferred,’ ‘desired,’ or ‘desirable,’ and words ofsimilar meaning should not be understood as implying that certainfeatures are critical, essential, or even important to the structure orfunction of the invention, but instead as merely intended to highlightalternative or additional features that may or may not be utilized in aparticular embodiment of the invention. Likewise, a group of itemslinked with the conjunction ‘and’ should not be read as requiring thateach and every one of those items be present in the grouping, but rathershould be read as ‘and/or’ unless expressly stated otherwise. Similarly,a group of items linked with the conjunction ‘or’ should not be read asrequiring mutual exclusivity among that group, but rather should be readas ‘and/or’ unless expressly stated otherwise.

Where a range of values is provided, it is understood that the upper andlower limit, and each intervening value between the upper and lowerlimit of the range is encompassed within the embodiments.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity. The indefinite article ‘a’ or ‘an’ does not exclude aplurality. A single processor or other unit may fulfill the functions ofseveral items recited in the claims. The mere fact that certain measuresare recited in mutually different dependent claims does not indicatethat a combination of these measures cannot be used to advantage. Anyreference signs in the claims should not be construed as limiting thescope.

It will be further understood by those within the art that if a specificnumber of an introduced claim recitation is intended, such an intentwill be explicitly recited in the claim, and in the absence of suchrecitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases ‘at least one’ and ‘one or more’ to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles ‘a’ or ‘an’ limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases‘one or more’ or ‘at least one’ and indefinite articles such as ‘a’ or‘an’ (e.g., ‘a’ and/or ‘an’ should typically be interpreted to mean ‘atleast one’ or ‘one or more’); the same holds true for the use ofdefinite articles used to introduce claim recitations. In addition, evenif a specific number of an introduced claim recitation is explicitlyrecited, those skilled in the art will recognize that such recitationshould typically be interpreted to mean at least the recited number(e.g., the bare recitation of ‘two recitations,’ without othermodifiers, typically means at least two recitations, or two or morerecitations). Furthermore, in those instances where a conventionanalogous to ‘at least one of A, B, and C, etc.’ is used, in generalsuch a construction is intended in the sense one having skill in the artwould understand the convention (e.g., ‘a system having at least one ofA, B, and C’ would include but not be limited to systems that have Aalone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together, etc.). In those instances where aconvention analogous to ‘at least one of A, B, or C, etc.’ is used, ingeneral such a construction is intended in the sense one having skill inthe art would understand the convention (e.g., ‘a system having at leastone of A, B, or C’ would include but not be limited to systems that haveA alone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together, etc.). It will be furtherunderstood by those within the art that virtually any disjunctive wordand/or phrase presenting two or more alternative terms, whether in thedescription, claims, or drawings, should be understood to contemplatethe possibilities of including one of the terms, either of the terms, orboth terms. For example, the phrase ‘A or B’ will be understood toinclude the possibilities of ‘A’ or ‘B’ or ‘A and B.’

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification are to be understood as beingmodified in all instances by the term ‘about.’ Accordingly, unlessindicated to the contrary, the numerical parameters set forth herein areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of anyclaims in any application claiming priority to the present application,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

Furthermore, although the foregoing has been described in some detail byway of illustrations and examples for purposes of clarity andunderstanding, it is apparent to those skilled in the art that certainchanges and modifications may be practiced. Therefore, the descriptionand examples should not be construed as limiting the scope of theinvention to the specific embodiments and examples described herein, butrather to also cover all modification and alternatives coming with thetrue scope and spirit of the invention.

What is claimed is:
 1. A topical composition for promoting skinrestoration, comprising: a tripeptide-1 present at 1-10 ppm and one ormore peptides selected from a dipeptide, a tripeptide, or atetrapeptide; and a hexpeptide-12 present at 1-10 ppm and a hexapeptidewith amino acid sequence different from the hexapeptide-12; wherein thetopical composition promotes restoration of damaged or aging skin. 2.The topical composition of claim 1, wherein the composition is aqueous.3. The topical composition of claim 1, wherein the hexapeptide withamino acid sequence different from the hexapeptide-12 is present at 1-10ppm.
 4. The topical composition of claim 1, wherein the one or morepeptides comprise a tripeptide with amino acid sequence different fromtripeptide-1.
 5. The topical composition of claim 1, wherein the one ormore peptides comprise a dipeptide.
 6. The topical composition of claim1, further comprising one or more additional ingredients selected fromthe group consisting of disodium EDTA, niacinamide, caprylyl glycol,caprylhydroxamic acid, glycerin, phenoxyethanol, ethylhexylglycerin,betaine, propanediol, phospholipids, isopropyl palmitate, lecithin,polyacrylate-13, polysorbate 20, Squalane, Dunaliella Salina extract,phytosterols, Olea Europaea fruit oil, hydrolyzed pea protein,Butyrospermum Parkii (Shea) Butter, ceramide NP, tocopherol, butyleneglycol, caprylyl methicone, ascorbyl palmitate, phosphatidylserine, orcombinations thereof.
 7. The topical composition of claim 1, wherein thetopical composition further comprises Olea Europaea fruit oil present atno more than 0.050 wt. %.
 8. The topical composition of claim 1, whereinthe topical composition further comprises phosphatidylserine present at0.01-5.0 wt. %.
 9. A topical composition for promoting skin restoration,comprising: a tripeptide-1 present at 1-10 ppm; and a hexapeptide-12present at 1-10 ppm, wherein the topical composition promotesrestoration of damaged or aging skin.
 10. The topical composition ofclaim 9, wherein the tripeptide-1 comprises palmitoyl tripeptide-1,myristoyl tripeptide-1, or a combination thereof.
 11. The topicalcomposition of claim 9, wherein the hexapeptide-12 comprises palmitoylhexapeptide-12, myristoyl hexapeptide-12, or a combination thereof. 12.A topical composition for promoting skin restoration, comprising: atripeptide-1 present at 1-10 ppm and a tetrapeptide; and ahexapeptide-12 present at 1-10 ppm and a hexapeptide with amino acidsequence different from the hexapeptide-12; wherein the topicalcomposition promotes restoration of damaged or aging skin.
 13. Thetopical composition of claim 12, wherein the tetrapeptide is present at1-10 ppm.